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Sample records for ligase gp78 increases

  1. Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of {alpha}-1-antitrypsin

    SciTech Connect

    Shen Yuxian; Ballar, Petek; Fang, Shengyun . E-mail: fangs@umbi.umd.edu

    2006-11-03

    Deficiency of circulating {alpha}-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

  2. p38 MAP kinase-dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER-mitochondria association and mitochondria motility.

    PubMed

    Li, Lei; Gao, Guang; Shankar, Jay; Joshi, Bharat; Foster, Leonard J; Nabi, Ivan R

    2015-11-01

    Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.

  3. Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases.

    PubMed

    Wang, Ying; Ha, Seung-Wook; Zhang, Tianpeng; Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

    2014-04-30

    gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.

  4. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain.

  5. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain. PMID:22045301

  6. Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase.

    PubMed

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

    2013-08-01

    Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K- and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42- and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1- and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics.

  7. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    PubMed

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  8. The complex biology of autocrine motility factor/phosphoglucose isomerase (AMF/PGI) and its receptor, the gp78/AMFR E3 ubiquitin ligase.

    PubMed

    Fairbank, Maria; St-Pierre, Pascal; Nabi, Ivan R

    2009-08-01

    Phosphoglucose isomerase (PGI) is a glycolytic enzyme that exhibits a dual function as an extracellular cytokine, under the name autocrine motility factor (AMF). Its cell surface receptor, gp78/AMFR, is also localized to the endoplasmic reticulum where it functions as an E3 ubiquitin ligase. Expression of both AMF/PGI and gp78/AMFR is associated with cancer and, in this review, we will discuss various aspects of the biology of this ligand-receptor complex and its role in tumor progression.

  9. gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum

    PubMed Central

    Zhang, Ting; Xu, Yue; Liu, Yanfen; Ye, Yihong

    2015-01-01

    Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA–mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation. PMID:26424800

  10. AMF/G6PI induces differentiation of leukemic cells via an unknown receptor that differs from gp78.

    PubMed

    Haga, Arayo; Komazaki, Sachiko; Funasaka, Tatsuyoshi; Hashimoto, Kazunori; Yokoyama, Yuichi; Watanabe, Hideomi; Raz, Avraham; Nagase, Hisamitsu

    2006-10-01

    Autocrine Motility Factor (AMF)/maturation factor (MF)/neuroleukin (NLK) is a multifunctional protein, which acts as a glucose 6-phosphate isomerase (G6PI) intracellularly. Exto-G6PI stimulates invasion and metastasis of tumor cells, neurotropic growth and differentiation of leukemic cells. The cell motility and proliferation receptor is known to be gp78 (78 kilo-Dalton glycoprotein), which has seven transmembrane domains in its N-terminal region, but the maturation factor receptor remains unclear. The human acute monocytic leukemia line does not express gp78 and its motile activity is not enhanced by AMF though it is well differentiated by AMF exposure. The forced expression of gp78 in leukemic cells recovered acceptable motile stimulation, concomitant with reduced differentiation ability. Two unknown proteins were detected by crosslinking between AMF and leukemic cells. The results of this report suggest that the receptor molecule for AMF/NLK/MF in leukemic differentiation is not gp78.

  11. TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation

    PubMed Central

    Weng, Pei-Lun; Yeh, Te-Huei

    2016-01-01

    Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice. This finding shows the functional significance of TSGΔ154-1054 in preventing the ubiquitin-proteasome proteolysis of TSG101, which increases tumor malignancy and hints at its potential as a therapeutic target in cancer treatment. PMID:26811492

  12. The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2) Is Increased in Human Lung Cancer

    PubMed Central

    Moreno, Paula; Lara-Chica, Maribel; Soler-Torronteras, Rafael; Caro, Teresa; Medina, Manuel; Álvarez, Antonio; Salvatierra, Ángel; Muñoz, Eduardo; Calzado, Marco A.

    2015-01-01

    Objectives Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH) proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry) in human non-small cell lung cancer (NSCLC) samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features. Materials and Methods One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables. Results The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry) in non-small cell lung cancer (NSCLC). We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC) and squamous cell lung cancer (SCC). Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(18F)fluoro-D-glucose) uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates

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

    PubMed

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

    2013-07-19

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

  14. SCF ubiquitin ligase targeted therapies

    PubMed Central

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

    2015-01-01

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

  15. Tubulin-tyrosine Ligase (TTL)-mediated Increase in Tyrosinated α-Tubulin in Injured Axons Is Required for Retrograde Injury Signaling and Axon Regeneration.

    PubMed

    Song, Wenjun; Cho, Yongcheol; Watt, Dana; Cavalli, Valeria

    2015-06-01

    Injured peripheral neurons successfully activate a pro-regenerative program to enable axon regeneration and functional recovery. The microtubule-dependent retrograde transport of injury signals from the lesion site in the axon back to the cell soma stimulates the increased growth capacity of injured neurons. However, the mechanisms initiating this retrograde transport remain poorly understood. Here we show that tubulin-tyrosine ligase (TTL) is required to increase the levels of tyrosinated α-tubulin at the axon injury site and plays an important role in injury signaling. Preventing the injury-induced increase in tyrosinated α-tubulin by knocking down TTL impairs retrograde organelle transport and delays activation of the pro-regenerative transcription factor c-Jun. In the absence of TTL, axon regeneration is reduced severely. We propose a model in which TTL increases the levels of tyrosinated α-tubulin locally at the injury site to facilitate the retrograde transport of injury signals that are required to activate a pro-regenerative program.

  16. Hyperthermic stress-induced increase in the expression of glutamate-cysteine ligase and glutathione levels in the symbiotic sea anemone Aiptasia pallida.

    PubMed

    Sunagawa, Shinichi; Choi, Jinah; Forman, Henry Jay; Medina, Mónica

    2008-09-01

    Hyperthermic stress is known to trigger the loss of unicellular algae from a number of symbiotic cnidarians, a phenomenon commonly referred to as bleaching. Oxidative and nitrosative stress have been suggested to play a major role during the process of bleaching, however the underlying molecular mechanisms are still poorly understood. In animals, the intracellular tripeptide glutathione (GSH) is involved in antioxidant defense, redox homeostasis and intracellular redox signaling. Therefore, we tested the hypothesis that hyperthermal stress-induced bleaching in Aiptasia pallida, a model for symbiotic cnidarians, results in increased levels of GSH synthesis. We report the cDNA sequence and functional analysis of the catalytic subunit of glutamate-cysteine ligase (GCLC), which catalyzes the rate-limiting step in GSH biosynthesis. In a time-series experiment, both GCLC gene expression and total GSH levels increased 4- and 1.5-fold, respectively, in response to hyperthermal stress. These results suggest that hyperthermal stress triggers adaptive increases in intracellular GSH biosynthesis in cnidarians as a protective response to oxidative/nitrosative stress. Our results show the conserved function of GCLC and GSH across animals while placing a new perspective on the role of GSH in redox signaling during cnidarian bleaching. PMID:18602489

  17. [Ligase chain reaction (LCR)].

    PubMed

    Yamanishi, K; Yasuno, H

    1993-06-01

    Ligase chain reaction (LCR) is a ligation-mediated amplification technique of a target DNA sequence using oligonucleotides and thermostable ligase. LCR is useful for the detection of known DNA sequences and point mutations in a limited amount of DNA. We introduce the principle, development, and protocol of this simple and convenient technique for DNA analysis.

  18. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  19. Distinct mechanisms controlling rough and smooth endoplasmic reticulum contacts with mitochondria.

    PubMed

    Wang, Peter T C; Garcin, Pierre O; Fu, Min; Masoudi, Matthew; St-Pierre, Pascal; Panté, Nelly; Nabi, Ivan R

    2015-08-01

    Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

  20. Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at adulthood.

    PubMed

    Chutabhakdikul, Naunchan; Surakul, Pornprom

    2013-11-01

    Exposure to excessive glucocorticoids during fetal development period contributes to later life psychopathology. Prenatal stress decreases dendritic spine density and impair LTP in the hippocampus of rat pups, however, the mechanisms regulating these changes are still unclear. Glutamate receptors are localized in the postsynaptic density. PSD-95 is a postsynaptic scaffolding protein that plays a role in synaptic maturation and regulation of the synaptic strength and plasticity. PSD-95 interacts with other proteins to form the protein networks that promote dendritic spine formation. The present study investigated the effect of prenatal stress on the levels of scaffolding proteins of NMDA receptor in the hippocampus in order to explain how prenatal stress alters the amount of NMDA receptor in the pups' brain. Pregnant rats were randomly assigned to either the prenatal stress (PS) or the control group (C). The pregnant rats in the PS group were restrained in a plexiglas restrainer for 4h/day during the GD 14-21. Control rats were left undisturbed for the duration of their pregnancies. The amount of PSD-95, SPAR, NR2A and NR2B, as well as the levels of Snk Polo-like kinase 2 and the SCF β-TrCP ubiquitin ligase were measured in the hippocampus of the offspring. The results show that prenatal stress induces a reduction in the amount of NR2B and NR2A subunits in the hippocampus of rat pups, parallel to the decrease in PSD-95 and SPAR at P40 and P60. Moreover, prenatal stress increases Snk and β-TrCP in the hippocampus of rat pups, and the timing correlates with the decrease of SPAR and PSD-95. Prenatal stress also induces a significantly increases in the level of ubiquitinated SPAR in the hippocampus of rat pups at adulthood. The results suggest that degradation of SPAR via UPS system may contribute to the loss of PSD-95 and NMDA receptor subunits in the hippocampus of rat pups at adulthood. In conclusion, the present work demonstrates that the developing brain is

  1. Tag Team Ubiquitin Ligases.

    PubMed

    Kleiger, Gary; Deshaies, Raymond

    2016-08-25

    Cullin-RING (CRL) and RING1-IBR-RING2 (RBR) are two distinct types of ubiquitin ligases. In this issue, Scott et al. show that CRLs activate the RBR enzyme ARIH1 to initiate ubiquitin chains on CRL substrates, thereby marking an unexpected and important advance in our understanding of both enzymes. PMID:27565338

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  3. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    SciTech Connect

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  4. DNA and RNA ligases: structural variations and shared mechanisms.

    PubMed

    Pascal, John M

    2008-02-01

    DNA and RNA ligases join 3' OH and 5' PO4 ends in polynucleotide substrates using a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the polynucleotide substrate with AMP. In the past three years, several polynucleotide ligases have been crystallized in complex with nucleic acid, providing the introductory views of ligase enzymes engaging their substrates. Crystal structures for two ATP-dependent DNA ligases, an NAD+-dependent DNA ligase, and an ATP-dependent RNA ligase demonstrate how ligases utilize the AMP group and their multi-domain architectures to manipulate nucleic acid structure and catalyze the end-joining reaction. Together with unliganded crystal structures of DNA and RNA ligases, a more comprehensive and dynamic understanding of the multi-step ligation reaction mechanism has emerged.

  5. DNA ligases in the repair and replication of DNA.

    PubMed

    Timson, D J; Singleton, M R; Wigley, D B

    2000-08-30

    DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA. Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.

  6. A purple acidophilic di-ferric DNA ligase from Ferroplasma.

    PubMed

    Ferrer, Manuel; Golyshina, Olga V; Beloqui, Ana; Böttger, Lars H; Andreu, José M; Polaina, Julio; De Lacey, Antonio L; Trautwein, Alfred X; Timmis, Kenneth N; Golyshin, Peter N

    2008-07-01

    We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two Fe(3+)-tyrosinate centers and lacks any requirement for either Mg(2+) or K(+) for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require Mg(2+)/K(+) for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the Fe(3+) to Fe(2+) results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH.

  7. Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

    PubMed Central

    Gore, Nigel R.; Wray, John L.

    1978-01-01

    Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-3H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [15N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [14N]nitrate and 100% H2O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation. PMID:16660229

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

    PubMed

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

    2003-08-01

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

  9. RBR E3-ligases at work.

    PubMed

    Smit, Judith J; Sixma, Titia K

    2014-02-01

    The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.

  10. Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

    PubMed

    Blondal, Thorarinn; Hjorleifsdottir, Sigridur H; Fridjonsson, Olafur F; Aevarsson, Arnthor; Skirnisdottir, Sigurlaug; Hermannsdottir, Anna Gudny; Hreggvidsson, Gudmundur O; Smith, Albert Vernon; Kristjansson, Jakob K

    2003-12-15

    Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.

  11. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    PubMed Central

    Anella, Fabrizio; Danelon, Christophe

    2014-01-01

    The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA) vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment. PMID:25513761

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

    PubMed

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

    2016-05-30

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

  13. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

    PubMed Central

    Lauer, G; Rudd, E A; McKay, D L; Ally, A; Ally, D; Backman, K C

    1991-01-01

    We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step. Images PMID:1840584

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

    PubMed Central

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

    2013-01-01

    Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein. PMID:23770887

  15. A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors.

    PubMed

    Jang, Jae-Woo; Lee, Won-Young; Lee, Jae-Ho; Moon, Sung-Hwan; Kim, Chang-Hoon; Chung, Hyung-Min

    2011-07-01

    Alterations in ubiquitin-proteasome system (UPS) have been implicated in the etiology of human cardiovascular diseases. Skp1/Cul1/F-box (SCF) ubiquitin E3 ligase complex plays a pivotal role in ubiquitination of cardiac proteins. However, a specific ubiquitin E3 ligase responsible for the destruction of cardiac transcription factors such as Nkx2-5, Isl1, Mef2C, and Tbx5 remains elusive to date. Here, we show that a novel F-box containing Fbxo25 is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 expression was nuclei-specific in vitro and cardiomyocytes. Expression level of Fbxo25 was higher in a fetal heart than an adult. Moreover, Fbxo25 expression was increased along with those of cardiac-specific genes during cardiomyocyte development from ESCs. Fbxo25 expression facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C. Especially, Fbxo25 ubiquitinated Nkx2-5, Isl1, and Hand1. Altogether, Fbxo25 acts as an ubiquitin E3 ligase to target cardiac transcription factors including Nkx2-5, Isl1, and Hand1, indicating that cardiac protein homeostasis through Fbxo25 has a pivotal impact on cardiac development.

  16. Chromosome demise in the wake of ligase-deficient replication

    PubMed Central

    Kouzminova, Elena A.; Kuzminov, Andrei

    2012-01-01

    Summary Bacterial DNA ligases, NAD+-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. PMID:22582878

  17. KF-1 Ubiquitin Ligase: An Anxiety Suppressor

    PubMed Central

    Hashimoto-Gotoh, Tamotsu; Iwabe, Naoyuki; Tsujimura, Atsushi; Takao, Keizo; Miyakawa, Tsuyoshi

    2009-01-01

    Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located on the endoplasmic reticulum (ER), may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. It is surmised that KF-1 degrades some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD). Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice may be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds. PMID:19753093

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

    PubMed

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

    2016-02-01

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

  19. Characterization of Agrobacterium tumefaciens DNA ligases C and D.

    PubMed

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3'-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3'-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3'-OH end. The PE domains also have a 3'-phosphatase activity on an all-DNA primer-template that yields a 3'-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.

  20. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    PubMed

    Arakawa, Hiroshi; Iliakis, George

    2015-01-01

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

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

    PubMed

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

    2016-01-01

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

  2. Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells.

    PubMed

    Stawiecka-Mirota, Marta; Kamińska, Joanna; Urban-Grimal, Daniele; Haines, Dale S; Zoładek, Teresa

    2008-11-01

    Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution. PMID:18804462

  3. Dysregulation of ubiquitin ligases in cancer

    PubMed Central

    Ronai, Ze’ev A.

    2015-01-01

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

  4. Heterologous expression of rice SUMO E3 ligase (OsSIZ1) enhances drought and heat tolerance in transgenic cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis gene AtSIZ1 encodes a SUMO E3 ligase that plays important roles in plant response to abiotic stresses such as drought, heat, cold, salt, and nutrient starvation. Loss of function in AtSIZ1 leads to increased sensitivity to drought, heat, and salt stresses, whereas overexpression of t...

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

    PubMed Central

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

    2012-01-01

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

  6. Characterization of Agrobacterium tumefaciens DNA ligases C and D

    PubMed Central

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ. PMID:17488851

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

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes. PMID:26362128

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

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

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

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

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

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

  10. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  11. The Hypoxia-controlled FBXL14 Ubiquitin Ligase Targets SNAIL1 for Proteasome Degradation*

    PubMed Central

    Viñas-Castells, Rosa; Beltran, Manuel; Valls, Gabriela; Gómez, Irene; García, José Miguel; Montserrat-Sentís, Bàrbara; Baulida, Josep; Bonilla, Félix; de Herreros, Antonio García; Díaz, Víctor M.

    2010-01-01

    The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase β-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3β. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3β, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3β. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia. PMID:19955572

  12. Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

    PubMed Central

    Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

    2016-01-01

    Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

  13. Mapping L1 Ligase ribozyme conformational switch

    PubMed Central

    Giambaşu, George M.; Lee, Tai-Sung; Scott, William G.; York, Darrin M.

    2012-01-01

    L1 Ligase (L1L)molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5’-to-3’ phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA World hypothesis. L1L crystal structure captures two distinct conformations that differ by a re-orientation of one of the stems by around 80 Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution, and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a 3-state/2-step process. The first step involves a large-amplitude swing that re-orients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network. PMID:22771572

  14. Genes of succinyl-CoA ligase from Saccharomyces cerevisiae.

    PubMed

    Przybyla-Zawislak, B; Dennis, R A; Zakharkin, S O; McCammon, M T

    1998-12-01

    Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  16. Differential dependence on DNA ligase of type II restriction enzymes: a practical way toward ligase-free DNA automaton.

    PubMed

    Chen, Peng; Li, Jing; Zhao, Jian; He, Lin; Zhang, Zhizhou

    2007-02-16

    DNA computing study is a new paradigm in computer science and biological computing fields. As one of DNA computing approaches, DNA automaton is composed of the hardware, input DNA molecule and state transition molecules. By now restriction enzymes are key hardware for DNA computing automaton. It has been found that DNA computing efficiency may be independent on DNA ligases when type IIS restriction enzymes like FokI are used as hardware. In this study, we compared FokI with four other distinct enzymes HgaI, BsmFI, BbsI, and BseMII, and found their differential independence on T4 DNA ligase when performing automaton reactions. Since DNA automaton is a potential powerful tool to tackle gene relationship in genomic network scale, the feasible ligase-free DNA automaton may set an initial base to develop functional DNA automata for various DNA technology development and implications in genetics study in the near future.

  17. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    PubMed Central

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR. PMID:23967291

  18. Role of ubiquitin ligases in neural stem and progenitor cells.

    PubMed

    Naujokat, Cord

    2009-01-01

    Ubiquitin ligases are central components of the ubiquitin-proteasome system (UPS), the major machinery for regulated proteolysis in eukaryotic cells. Proteins essential for regulating development, differentiation, proliferation, cell cycling, apoptosis, gene transcription, and signal transduction undergo posttranslational processing via selection by ubiquitin ligases and subsequent controlled proteolysis by the 26S proteasome, the proteolytic unit of the UPS. Neural stem cells (NSCs) are self-renewing multipotent cells of the embryonic and adult mammalian central nervous system. In the last few years, NSCs have generated considerable interest because of their potential to repair neurological damage in preclinical models of stroke, spinal cord injury, and neurodegenerative disease. Recent evidence reveals a central role of ubiquitin ligases in controlling the development, survival, differentiation, and programming of neural stem and progenitor cells. Here the current knowledge of the role and function of ubiquitin ligases in neural stem and progenitor cells is reviewed and insight into an important mechanism of NSC homeostasis by regulated proteolysis is provided. PMID:19479207

  19. The Role of Ubiquitin Ligases in Cardiac Disease

    PubMed Central

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

    2014-01-01

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

  20. CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory.

    PubMed

    Chen, Yan-Chu; Hsu, Wei-Lun; Ma, Yun-Li; Tai, Derek J C; Lee, Eminy H Y

    2014-07-16

    cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

  1. A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells

    SciTech Connect

    Petrini, J.H.J.; Huwiler, K.G.; Weaver, D.T. )

    1991-09-01

    Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells. DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anomalously dimeric form. To assess the role of DNA ligase function in the etiology of BS, the authors have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces pombe DNA ligase genes. Human DNA ligase I cDNAs from normal and BS cells complemented a S. cerevisiae DNA ligase mutation, and protein extracts prepared from S. cerevisiae transformants expressing normal and BS cDNA contained comparable levels of DNA ligase I activity. DNA sequencing and Northern blot analysis of DNA ligase I expression in two BS human fibroblast lines representing each of the two aberrant DNA ligase I molecular phenotypes demonstrated that this gene was unchanged in BS cells. Thus, another factor may be responsible for the observed reduction in DNA ligase I activity associated with this chromosomal breakage syndrome.

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

    PubMed Central

    Rodrigues, Elizabeth M.; Scudder, Samantha L.; Goo, Marisa S.

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. SIGNIFICANCE STATEMENT Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. PMID:26843640

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

    PubMed

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

    2010-02-01

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

  4. A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer.

    PubMed

    Satija, Yatendra Kumar; Bhardwaj, Abhishek; Das, Sanjeev

    2013-12-15

    E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) are the key components of ubiquitin proteasome system which plays a critical role in cellular protein homeostasis. Any shortcoming in their biological roles can lead to various diseases including cancer. The dynamic interplay between ubiquitylation and deubiquitylation determines the level and activity of several proteins including p53, which is crucial for cellular stress response and tumor suppression pathways. In this review, we describe the different types of E3 ubiquitin ligases including those targeting tumor suppressor p53, SCF ligases and RING type ligases and accentuate on biological functions of few important E3 ligases in the cellular regulatory networks. Tumor suppressor p53 level is tightly regulated by multiple E3 ligases including Mdm2, COP1, Pirh2, etc. SCF ubiquitin ligase complexes are key regulators of cell cycle and signal transduction. BRCA1 and VHL RING type ligases function as tumor suppressors and play an important role in DNA repair and hypoxia response respectively. Further, we discuss the biological consequences of deregulation of the E3 ligases and the implications for cancer development. We also describe deubiquitylases which reverse the process of ubiquitylation and regulate diverse cellular pathways including metabolism, cell cycle control and chromatin remodelling. As the E3 ubiquitin ligases and DUBs work in a substrate specific manner, an improved understanding of them can lead to better therapeutics for cancer.

  5. Parkin and relatives: the RBR family of ubiquitin ligases.

    PubMed

    Marín, Ignacio; Lucas, J Ignasi; Gradilla, Ana-Citlali; Ferrús, Alberto

    2004-05-19

    Mutations in the parkin gene cause autosomal-recessive juvenile parkinsonism. Parkin encodes a ubiquitin-protein ligase characterized by having the RBR domain, composed of two RING fingers plus an IBR/DRIL domain. The RBR family is defined as the group of genes whose products contain an RBR domain. RBR family members exist in all eukaryotic species for which significant sequence data is available, including animals, plants, fungi, and several protists. The integration of comparative genomics with structural and functional data allows us to conclude that RBR proteins have multiple roles, not only in protein quality control mechanisms, but also as indirect regulators of transcription. A recently formulated hypothesis, based on a case of gene fusion, suggested that RBR proteins may be often part of cullin-containing ubiquitin ligase complexes. Recent data on Parkin protein agrees with that hypothesis. We discuss the involvement of RBR proteins in several neurodegenerative diseases and cancer.

  6. HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex*

    PubMed Central

    Wang, Xin; Singh, Shailbala; Jung, Hae-Yun; Yang, Guojun; Jun, Sohee; Sastry, K. Jagannadha; Park, Jae-Il

    2013-01-01

    Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis. PMID:23612978

  7. Kinetic framework for ligation by an efficient RNA ligase ribozyme.

    PubMed

    Bergman, N H; Johnston, W K; Bartel, D P

    2000-03-21

    The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches. PMID:10715133

  8. The Kaposi's sarcoma-associated herpesvirus K5 E3 ubiquitin ligase modulates targets by multiple molecular mechanisms.

    PubMed

    Means, Robert E; Lang, Sabine M; Jung, Jae U

    2007-06-01

    Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms.

  9. The Kaposi's Sarcoma-Associated Herpesvirus K5 E3 Ubiquitin Ligase Modulates Targets by Multiple Molecular Mechanisms▿

    PubMed Central

    Means, Robert E.; Lang, Sabine M.; Jung, Jae U.

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms. PMID:17409151

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

    PubMed Central

    Bornstein, Gil; Ganoth, Dvora; Hershko, Avram

    2006-01-01

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

  11. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  12. The relationship between polymorphisms in the glutamate cysteine ligase gene and asthma susceptibility.

    PubMed

    Polonikov, A V; Ivanov, V P; Solodilova, M A; Khoroshaya, I V; Kozhuhov, M A; Panfilov, V I

    2007-11-01

    The present study was designed to investigate an association of common -588C/T and -23G/T polymorphisms within glutamate cysteine ligase modifier subunit gene with susceptibility to bronchial asthma. A total of 435 ethnically Russian subjects were recruited in this study, including 221 patients with asthma and 214 sex and age matched healthy subjects. As previously reported, the -588C/T and -23G/T polymorphisms were completely linked. The -588TT/-23TT genotype was found to be associated with decreased risk of allergic asthma after adjustment for age, gender and smoking status using multivariate logistic regression analysis (OR=0.33 95% CI 0.15-0.70, p=0.036). However, the -588CT/-23GT genotype was associated with increased risk of non-allergic asthma (OR=2.03 95% CI 1.05-3.90, p=0.06). This is a first study reporting the association between genetic variations in the glutamate cysteine ligase gene and susceptibility to bronchial asthma. PMID:17643973

  13. [The applications of thermostable ligase chain reaction in facilitating DNA recombination].

    PubMed

    Xiangda, Zhou; Xiao, Song; Cong, Huai; Haiyan, Sun; Hongyan, Chen; Daru, Lu

    2016-02-01

    The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

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

    PubMed

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

    2015-12-01

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

  15. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  16. Degradation of host ubiquitin E3 ligase Itch by human cytomegalovirus UL42.

    PubMed

    Koshizuka, Tetsuo; Tanaka, Keiichiro; Suzutani, Tatsuo

    2016-01-01

    Human cytomegalovirus (HCMV) UL42 is classified as a CMV-specific but function-unknown gene. According to its amino acid sequence, UL42 has a C-terminal hydrophobic domain predicted to be a transmembrane domain and two PPxY (PY) motifs in its N terminus, but no N-terminal signal peptide. These features resemble those of herpes simplex virus (HSV) UL56 and varicella-zoster virus ORF0. HCMV UL42 interacts with Itch, a member of the Nedd4 family of ubiquitin E3 ligases, through its PY motifs as observed in HSV UL56. HCMV UL42 was partially colocalized with the trans-Golgi network and cytoplasmic vesicles in transfected fibroblasts. Itch was colocalized with HCMV UL42 and accumulated in a fine-speckled pattern in the cytoplasm. UL42 induced the ubiquitination and degradation of Itch in HCMV-infected fibroblasts, and was partially colocalized with p62, a ubiquitin-binding protein, and CD63, a marker of lysosome and multivesicular bodies. The electrophoretic pattern of Itch was altered by infection with HCMV and the amount of Itch was increased by the deletion of UL42. Our findings suggest that the regulatory function of the Nedd4 E3 ligase family and the structural features of HCMV UL42 are conserved characteristics in herpesviruses. PMID:26555021

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

    PubMed Central

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

    2015-01-01

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

  18. [Cloning and tissue expression of 4-coumarate coenzyme A ligase gene in Angelica sinensis].

    PubMed

    Wen, Sui-chao; Wang, Yin-quan; Luo, Jun; Xia, Qi; Fan, Qin; Li, Shu-nan; Wang, Zhen-heng

    2015-12-01

    4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis. PMID:27245029

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

    PubMed Central

    Lin, Xiao-Li; Niu, De; Hu, Zi-Liang; Kim, Dae Heon; Jin, Yin Hua; Cai, Bin; Liu, Peng; Miura, Kenji; Yun, Dae-Jin; Kim, Woe-Yeon; Lin, Rongcheng

    2016-01-01

    COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants. PMID:27128446

  20. An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis[W

    PubMed Central

    Huang, Yan; Minaker, Sean; Roth, Charlotte; Huang, Shuai; Hieter, Philip; Lipka, Volker; Wiermer, Marcel; Li, Xin

    2014-01-01

    Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway. PMID:24449689

  1. Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

    PubMed Central

    Henke, Sarah K.; Cronan, John E.

    2014-01-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

  2. Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

    PubMed

    Gaid, Mariam M; Sircar, Debabrata; Müller, Andreas; Beuerle, Till; Liu, Benye; Ernst, Ludger; Hänsch, Robert; Beerhues, Ludger

    2012-11-01

    Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

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

    PubMed Central

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

    2016-01-01

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

  4. Defining interactions between DNA-PK and ligase IV/XRCC4

    SciTech Connect

    Hsu, Hsin-Ling; Yannone, Steven M.; Chen, David J.

    2001-04-10

    Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.

  5. 4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements

    NASA Technical Reports Server (NTRS)

    Church, D. L.; Galston, A. W.

    1988-01-01

    When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

  6. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis.

    PubMed

    Zhang, M; Zhu, X; Zhang, Y; Cai, Y; Chen, J; Sivaprakasam, S; Gurav, A; Pi, W; Makala, L; Wu, J; Pace, B; Tuan-Lo, D; Ganapathy, V; Singh, N; Li, H

    2015-12-01

    The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. PMID:25952549

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

    PubMed Central

    Aleidi, Shereen M.; Howe, Vicky; Sharpe, Laura J.; Yang, Alryel; Rao, Geetha; Brown, Andrew J.; Gelissen, Ingrid C.

    2015-01-01

    The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. PMID:26296893

  8. Rapid Assembly of DNA via Ligase Cycling Reaction (LCR).

    PubMed

    Chandran, Sunil

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the ligase chain reaction (LCR). The LCR method utilizes non-overlapping DNA parts that are ligated together with the guidance of bridging oligos. Using this method, we have successfully assembled up to 20 DNA parts in a single reaction or DNA constructs up to 26 kb in size. PMID:27671935

  9. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Wang, Julia; Jennings, Alexandra K.

    2016-01-01

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

  12. Regulation of ubiquitin ligase dynamics by the nucleolus

    PubMed Central

    Mekhail, Karim; Khacho, Mireille; Carrigan, Amanda; Hache, Robert R.J.; Gunaratnam, Lakshman; Lee, Stephen

    2005-01-01

    Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's β-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state. PMID:16129783

  13. The antiobesity factor WDTC1 suppresses adipogenesis via the CRL4WDTC1 E3 ligase.

    PubMed

    Groh, Beezly S; Yan, Feng; Smith, Matthew D; Yu, Yanbao; Chen, Xian; Xiong, Yue

    2016-05-01

    WDTC1/Adp encodes an evolutionarily conserved suppressor of lipid accumulation. While reduced WDTC1 expression is associated with obesity in mice and humans, its cellular function is unknown. Here, we demonstrate that WDTC1 is a component of a DDB1-CUL4-ROC1 (CRL4) E3 ligase. Using 3T3-L1 cell culture model of adipogenesis, we show that disrupting the interaction between WDTC1 and DDB1 leads to a loss of adipogenic suppression by WDTC1, increased triglyceride accumulation and adipogenic gene expression. We show that the CRL4(WDTC) (1) complex promotes histone H2AK119 monoubiquitylation, thus suggesting a role for this complex in transcriptional repression during adipogenesis. Our results identify a biochemical role for WDTC1 and extend the functional range of the CRL4 complex to the suppression of fat accumulation. PMID:27113764

  14. Further analysis of XBAT32, an Arabidopsis RING E3 ligase, involved in ethylene biosynthesis.

    PubMed

    Prasad, Madhulika E; Stone, Sophia L

    2010-11-01

    The Arabidopsis RING E3 ligase, XBAT32, was previously characterized as a regulator of lateral root initiation. However, how XBAT32 function to modulate lateral root initiation was unknown. In our recent paper, we demonstrated that XBAT32 is involved in ethylene biosynthesis and it is through this function that XBAT32 is able to regulate lateral root production. Here we discuss a few other findings, observed in the ethylene overproducing mutant, xbat32, that reflect the effect of elevated ethylene levels on plant growth and development. Ethylene signaling also regulates plant responses to adverse environmental conditions such as high salinity. Consistent with ethylene's role as a stress hormone, xbat32 exhibited increased sensitivity to salt stress during seed germination and postgerminative growth. Thus, XBAT32 may also play a role in ethylene mediated response to abiotic stresses.

  15. Using molecular beacon to monitor activity of E. coli DNA ligase.

    PubMed

    Liu, Lingfeng; Tang, Zhiwen; Wang, Kemin; Tan, Weihong; Li, Jun; Guo, Qiuping; Meng, Xiangxian; Ma, Changbei

    2005-03-01

    NAD(+)-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD(+)-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 x 10(-4) Weiss Unit mL(-1) to 0.4 Weiss Unit mL(-1) and the detection limit of 4.0 x 10(-4) Weiss Unit mL(-1).

  16. DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells

    PubMed Central

    Oh, Sehyun; Harvey, Adam; Zimbric, Jacob; Wang, Yongbao; Nguyen, Thanh; Jackson, Pauline J.; Hendrickson, Eric A.

    2014-01-01

    Ku-dependent C-NHEJ (classic non-homologous end joining) is the primary DNA EJing (end joining) repair pathway in mammals. Recently, an additional EJing repair pathway (A-NHEJ; alternative-NHEJ) has been described. Currently, the mechanism of A-NHEJ is obscure although a dependency on LIGIII (DNA ligase III) is often implicated. To test the requirement for LIGIII in A-NHEJ we constructed a LIGIII conditionally-null human cell line using gene targeting. Nuclear EJing activity appeared unaffected by a deficiency in LIGIII as, surprisingly, so were random gene targeting integration events. In contrast, LIGIII was required for mitochondrial function and this defined the gene’s essential activity. Human Ku:LIGIII and Ku:LIGIV (DNA ligase IV) double knockout cell lines, however, demonstrated that LIGIII is required for the enhanced A-NHEJ activity that is observed in Ku-deficient cells. Most unexpectedly, however, the majority of EJing events remained LIGIV-dependent. In conclusion, although human LIGIII has an essential function in mitochondrial maintenance, it is dispensable for most types of nuclear DSB repair, except for the A-NHEJ events that are normally suppressed by Ku. Moreover, we describe that a robust Ku-independent, LIGIV-dependent repair pathway exists in human somatic cells. PMID:24837021

  17. Transcript profiling of jasmonate-elicited Taxus cells reveals a β-phenylalanine-CoA ligase.

    PubMed

    Ramírez-Estrada, Karla; Altabella, Teresa; Onrubia, Miriam; Moyano, Elisabeth; Notredame, Cedric; Osuna, Lidia; Vanden Bossche, Robin; Goossens, Alain; Cusido, Rosa M; Palazon, Javier

    2016-01-01

    Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

  18. Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases.

    PubMed

    Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A

    2016-07-01

    PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability.

  19. Human DNA ligase I cDNA: Cloning and functional expression in Saccharomyces cerevisiae

    SciTech Connect

    Barnes, D.E.; Kodama, Kenichi; Tomkinson, A.E.; Lindahl, T.; Lasko, D.D. ); Johnston, L.H. )

    1990-09-01

    Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomuces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

  20. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

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

  1. Human ITCH E3 ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease.

    PubMed

    Lohr, Naomi J; Molleston, Jean P; Strauss, Kevin A; Torres-Martinez, Wilfredo; Sherman, Eric A; Squires, Robert H; Rider, Nicholas L; Chikwava, Kudakwashe R; Cummings, Oscar W; Morton, D Holmes; Puffenberger, Erik G

    2010-03-12

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  2. Human ITCH E3 Ubiquitin Ligase Deficiency Causes Syndromic Multisystem Autoimmune Disease

    PubMed Central

    Lohr, Naomi J.; Molleston, Jean P.; Strauss, Kevin A.; Torres-Martinez, Wilfredo; Sherman, Eric A.; Squires, Robert H.; Rider, Nicholas L.; Chikwava, Kudakwashe R.; Cummings, Oscar W.; Morton, D. Holmes; Puffenberger, Erik G.

    2010-01-01

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  3. Structural Basis for Nick Recognition by a Minimal Pluripotent DNA Ligase

    SciTech Connect

    Nair,P.; Nandakumar, J.; Smith, P.; Odell, M.; Lima, C.; Shuman, S.

    2007-01-01

    Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-{angstrom} crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO{sub 4} nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a {beta}-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO{sub 4} termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.

  4. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  5. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  6. The CUL3-KLHL18 ligase regulates mitotic entry and ubiquitylates Aurora-A.

    PubMed

    Moghe, Saili; Jiang, Fei; Miura, Yoshie; Cerny, Ronald L; Tsai, Ming-Ying; Furukawa, Manabu

    2012-02-15

    The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.

  7. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    PubMed Central

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  8. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  9. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  10. Phenolic metabolism in petunia tissues. IV. - Properties of p-coumarate : coenzyme A ligase isoenzymes.

    PubMed

    Ranjeva, R; Boudet, A M; Faggion, R

    1976-01-01

    Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.

  11. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  12. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  13. HOIL-1L Functions as the PKCζ Ubiquitin Ligase to Promote Lung Tumor Growth

    PubMed Central

    Queisser, Markus A.; Dada, Laura A.; Deiss-Yehiely, Nimrod; Angulo, Martin; Zhou, Guofei; Kouri, Fotini M.; Knab, Lawrence M.; Liu, Jing; Stegh, Alexander H.; DeCamp, Malcolm M.; Budinger, G. R. Scott; Chandel, Navdeep S.; Ciechanover, Aaron; Iwai, Kazuhiro

    2014-01-01

    Rationale: Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described. Objectives: To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth. Methods: The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1–interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. Measurements and Main Results: Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL

  14. RNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.

    PubMed Central

    Sano, H; Feix, G

    1975-01-01

    Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleotides are released before ligation can occur. As judged from its behaviour during the various purification steps and from some of its properties, the nicking activity seems to be different from known nicking enzymes. PMID:1101228

  15. Cullin3-KLHL25 ubiquitin ligase targets ACLY for degradation to inhibit lipid synthesis and tumor progression.

    PubMed

    Zhang, Cen; Liu, Juan; Huang, Grace; Zhao, Yuhan; Yue, Xuetian; Wu, Hao; Li, Jun; Zhu, Junlan; Shen, Zhiyuan; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

    2016-09-01

    Increased lipid synthesis is a key characteristic of many cancers that is critical for cancer progression. ATP-citrate lyase (ACLY), a key enzyme for lipid synthesis, is frequently overexpressed or activated in cancer to promote lipid synthesis and tumor progression. Cullin3 (CUL3), a core protein for the CUL3-RING ubiquitin ligase complex, has been reported to be a tumor suppressor and frequently down-regulated in lung cancer. Here, we found that CUL3 interacts with ACLY through its adaptor protein, KLHL25 (Kelch-like family member 25), to ubiquitinate and degrade ACLY in cells. Through negative regulation of ACLY, CUL3 inhibits lipid synthesis, cell proliferation, and xenograft tumor growth of lung cancer cells. Furthermore, ACLY inhibitor SB-204990 greatly abolishes the promoting effect of CUL3 down-regulation on lipid synthesis, cell proliferation, and tumor growth. Importantly, low CUL3 expression is associated with high ACLY expression and poor prognosis in human lung cancer. In summary, our results identify CUL3-KLHL25 ubiquitin ligase as a novel negative regulator for ACLY and lipid synthesis and demonstrate that decreased CUL3 expression is an important mechanism for increased ACLY expression and lipid synthesis in lung cancer. These results also reveal that negative regulation of ACLY and lipid synthesis is a novel and critical mechanism for CUL3 in tumor suppression. PMID:27664236

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

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Yount, Jacob S.

    2015-01-01

    Interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection. PMID:26263374

  17. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms

    PubMed Central

    Swift, Robert V.; Amaro, Rommie E.

    2009-01-01

    Background Members of the nucleotidyltransferase superfamily known as DNA and RNA ligases carry out the enzymatic process of polynucleotide ligation. These guardians of genomic integrity share a three-step ligation mechanism, as well as common core structural elements. Both DNA and RNA ligases have experienced a surge of recent interest as chemotherapeutic targets for the treatment of a range of diseases, including bacterial infection, cancer, and the diseases caused by the protozoan parasites known as trypanosomes. Objective In this review, we will focus on efforts targeting pathogenic microorganisms; specifically, bacterial NAD+-dependent DNA ligases, which are promising broad-spectrum antibiotic targets, and ATP-dependent RNA editing ligases from Trypanosoma brucei, the species responsible for the devastating neurodegenerative disease, African sleeping sickness. Conclusion High quality crystal structures of both NAD+-dependent DNA ligase and the Trypanosoma brucei RNA editing ligase have facilitated the development of a number of promising leads. For both targets, further progress will require surmounting permeability issues and improving selectivity and affinity. PMID:20354588

  18. Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

    PubMed Central

    Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart

    2015-01-01

    ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway—covalent ligase-(lysyl-Nζ)–AMP•Mn2+ intermediate; ligase•ATP•(Mn2+)2 Michaelis complex; and ligase•Mn2+ complex—highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound “catalytic” metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes. PMID:26512110

  19. Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

    PubMed Central

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2015-01-01

    The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO4 duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO4, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization. PMID:25740837

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

    PubMed Central

    Chen, Hsin-Yi; Chen, Ruey-Hwa

    2016-01-01

    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

  1. Activation of the E3 ubiquitin ligase Parkin.

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2016-10-30

    E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis. PMID:27480167

  3. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  4. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

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

    PubMed

    Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

    2015-10-01

    Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance.

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. Identification of Erwinia stewartii by a ligase chain reaction assay.

    PubMed Central

    Wilson, W J; Wiedmann, M; Dillard, H R; Batt, C A

    1994-01-01

    A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material. Images PMID:7509585

  9. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  10. The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

    PubMed Central

    Capoccia, Benjamin J.; Jin, Ramon U.; Kong, Young-Yun; Peek, Richard M.; Fassan, Matteo; Rugge, Massimo; Mills, Jason C.

    2013-01-01

    After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease. PMID:23478405

  11. Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium tuberculosis

    PubMed Central

    Duckworth, Benjamin P.; Geders, Todd W.; Tiwari, Divya; Boshoff, Helena I.; Sibbald, Paul A.; Barry, Clifton E.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2011-01-01

    SUMMARY The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent sub-nanomolar enzyme inhibition and antitubercular activity against multi- and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the anti-bacterial activity is MtBPL-dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray co-crystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development. PMID:22118677

  12. Suramin inhibits cullin-RING E3 ubiquitin ligases

    PubMed Central

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

    2016-01-01

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

  13. Biotin Protein Ligase Is a Target for New Antibacterials

    PubMed Central

    Feng, Jiage; Paparella, Ashleigh S.; Booker, Grant W.; Polyak, Steven W.; Abell, Andrew D.

    2016-01-01

    There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5′-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5′-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. PMID:27463729

  14. Biotin Protein Ligase Is a Target for New Antibacterials.

    PubMed

    Feng, Jiage; Paparella, Ashleigh S; Booker, Grant W; Polyak, Steven W; Abell, Andrew D

    2016-01-01

    There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5'-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5'-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5'-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. PMID:27463729

  15. Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus*

    PubMed Central

    Soares da Costa, Tatiana P.; Tieu, William; Yap, Min Y.; Pendini, Nicole R.; Polyak, Steven W.; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D.; Wallace, John C.; Wilce, Matthew C. J.; Booker, Grant W.; Abell, Andrew D.

    2012-01-01

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class. PMID:22437830

  16. UHRF2, another E3 ubiquitin ligase for p53

    SciTech Connect

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

    2012-09-07

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

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

    PubMed Central

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

    2014-01-01

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

  18. COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function through Its E3 Ubiquitin Ligase Activity

    PubMed Central

    Kim, Joo Y.; Jang, In-Cheol; Seo, Hak S.

    2016-01-01

    Ubiquitination and sumoylation are essential post-translational modifications that regulate growth and development processes in plants, including control of hormone signaling mechanisms and responses to stress. This study showed that COP1 (Constitutive photomorphogenic 1) regulated the activity of Arabidopsis E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 through its E3 ubiquitin ligase activity. Yeast two hybrid analysis demonstrated that COP1 and AtSIZ1 directly interacted with one another, and subcellular localization assays indicated that COP1 and AtSIZ1 co-localized in nuclear bodies. Analysis of ubiquitination showed that AtSIZ1 was polyubiquitinated by COP1. The AtSIZ1 level was higher in cop1-4 mutants than in wild-type seedlings under light or dark conditions, and overexpression of a dominant-negative (DN)-COP1 mutant led to a substantial increase in AtSIZ1 accumulation. In addition, under drought, cold, and high salt conditions, SUMO-conjugate levels were elevated in DN-COP1-overexpressing plants and cop1-4 mutant plants compared to wild-type plants. Taken together, our results indicate that COP1 controls responses to abiotic stress by modulation of AtSIZ1 levels and activity. PMID:27536318

  19. The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

    PubMed Central

    Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

    2015-01-01

    The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

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

    PubMed Central

    Bulatov, Emil; Ciulli, Alessio

    2015-01-01

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

  1. A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

    PubMed Central

    Loregger, Anke; Cook, Emma Claire Laura; Nelson, Jessica Kristin; Moeton, Martina; Sharpe, Laura Jane; Engberg, Susanna; Karimova, Madina; Lambert, Gilles; Brown, Andrew John

    2015-01-01

    Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. PMID:26527619

  2. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    PubMed Central

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

  3. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

    PubMed

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences.

  4. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

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

    PubMed

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

    2012-05-01

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

  6. COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function through Its E3 Ubiquitin Ligase Activity.

    PubMed

    Kim, Joo Y; Jang, In-Cheol; Seo, Hak S

    2016-01-01

    Ubiquitination and sumoylation are essential post-translational modifications that regulate growth and development processes in plants, including control of hormone signaling mechanisms and responses to stress. This study showed that COP1 (Constitutive photomorphogenic 1) regulated the activity of Arabidopsis E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 through its E3 ubiquitin ligase activity. Yeast two hybrid analysis demonstrated that COP1 and AtSIZ1 directly interacted with one another, and subcellular localization assays indicated that COP1 and AtSIZ1 co-localized in nuclear bodies. Analysis of ubiquitination showed that AtSIZ1 was polyubiquitinated by COP1. The AtSIZ1 level was higher in cop1-4 mutants than in wild-type seedlings under light or dark conditions, and overexpression of a dominant-negative (DN)-COP1 mutant led to a substantial increase in AtSIZ1 accumulation. In addition, under drought, cold, and high salt conditions, SUMO-conjugate levels were elevated in DN-COP1-overexpressing plants and cop1-4 mutant plants compared to wild-type plants. Taken together, our results indicate that COP1 controls responses to abiotic stress by modulation of AtSIZ1 levels and activity. PMID:27536318

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

    PubMed Central

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

    2014-01-01

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. PMID:24576094

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

    PubMed

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

    2014-03-15

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.

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

    PubMed

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

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

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

    PubMed Central

    Raychaudhuri, Sumana; Espenshade, Peter J.

    2015-01-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1–Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking. PMID:25918164

  11. Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

    PubMed Central

    Liddiard, Kate; Ruis, Brian; Takasugi, Taylor; Harvey, Adam; Ashelford, Kevin E.; Hendrickson, Eric A.; Baird, Duncan M.

    2016-01-01

    Telomeres shorten with each cell division and can ultimately become substrates for nonhomologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterized more than 1400 telomere fusion events at the single-molecule level, using a combination of high-throughput sequence analysis together with experimentally induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse nontelomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions. Fusion frequency was markedly increased in the absence of TP53 checkpoint control and significantly modulated by the cellular capacity for classical, versus alternative, nonhomologous end joining (NHEJ). We observed a striking reduction in inter-chromosomal fusion events in cells lacking DNA ligase 4, in contrast to a remarkably consistent profile of intra-chromosomal fusion in the context of multiple genetic knockouts, including DNA ligase 3 and 4 double-knockouts. We reveal distinct mutational signatures associated with classical NHEJ-mediated inter-chromosomal, as opposed to alternative NHEJ-mediated intra-chromosomal, telomere fusions and evidence for an unanticipated sufficiency of DNA ligase 1 for these intra-chromosomal events. Our findings have implications for mechanisms driving cancer genome evolution. PMID:26941250

  12. ABD1 is an Arabidopsis DCAF substrate receptor for CUL4-DDB1-based E3 ligases that acts as a negative regulator of abscisic acid signaling.

    PubMed

    Seo, Kyoung-In; Lee, Jae-Hoon; Nezames, Cynthia D; Zhong, Shangwei; Song, Eunyoung; Byun, Myung-Ok; Deng, Xing Wang

    2014-02-01

    Members of the DDB1-CUL4-associated factors (DCAFs) family directly bind to DAMAGED DNA BINDING PROTEIN1 (DDB1) and function as the substrate receptors in CULLIN4-based E3 (CUL4) ubiquitin ligases, which regulate the selective ubiquitination of proteins. Here, we describe a DCAF protein, ABD1 (for ABA-hypersensitive DCAF1), that negatively regulates abscisic acid (ABA) signaling in Arabidopsis thaliana. ABD1 interacts with DDB1 in vitro and in vivo, indicating that it likely functions as a CUL4 E3 ligase substrate receptor. ABD1 expression is induced by ABA, and mutations in ABD1 result in ABA- and NaCl-hypersensitive phenotypes. Loss of ABD1 leads to hyperinduction of ABA-responsive genes and higher accumulation of the ABA-responsive transcription factor ABA INSENSITIVE5 (ABI5), hypersensitivity to ABA during seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, ultimately, increased drought tolerance. ABD1 directly interacts with ABI5 in yeast two-hybrid assays and associates with ABI5 in vivo by coimmunoprecipitation, and the interaction was found in the nucleus by bimolecular fluorescence complementation. Furthermore, loss of ABD1 results in a retardation of ABI5 degradation by the 26S proteasome. Taken together, these data suggest that the DCAF-CUL4 E3 ubiquitin ligase assembled with ABD1 is a negative regulator of ABA responses by directly binding to and affecting the stability of ABI5 in the nucleus. PMID:24563203

  13. Global functional profiling of human ubiquitome identifies E3 ubiquitin ligase DCST1 as a novel negative regulator of Type-I interferon signaling

    PubMed Central

    Nair, Sajith; Bist, Pradeep; Dikshit, Neha; Krishnan, Manoj N

    2016-01-01

    Type I interferon (IFN-I) mediated innate immune response controls virus infections by inducing the expression of interferon stimulated genes (ISGs). Although ubiquitination plays key roles in immune signaling regulation, a human genome-wide understanding of the role of E3 ubiquitin ligases in interferon mediated ISG induction is lacking. Here, we report a genome-wide profiling of the effect of ectopic expression of 521 E3 ubiquitin ligases and substrate recognition subunits encoded in the human genome (which constitutes 84.4% of all ubiquitination related genes encoded in the human genome, hereafter termed Human Ubiquitome) on IFNβ mediated induction of interferon stimulated DNA response element (ISRE) driven reporter activity. We identified 96 and 42 genes of the human ubiquitome as novel negative and positive regulators of interferon signaling respectively. Furthermore, we characterized DCST1 as a novel E3 ubiquitin ligase negatively regulating interferon response. Ectopic expression and gene silencing of DCST1 respectively attenuated and increased ISRE reporter activity. DCST1 regulated Type I interferon signaling by interacting with and promoting ubiquitination-mediated degradation of STAT2, an essential component of antiviral gene induction. In summary, this study provided a systems level view on the role of human ubiquitination associated genes in Type I interferon response. PMID:27782195

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

    PubMed

    Duplan, Vincent; Rivas, Susana

    2014-01-01

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

  15. Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

    PubMed

    Ogasawara, Yasushi; Kawata, Junpei; Noike, Motoyoshi; Satoh, Yasuharu; Furihata, Kazuo; Dairi, Tohru

    2016-06-17

    We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.

  16. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    SciTech Connect

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  17. Regulation of neuronal survival and morphology by the E3 ubiquitin ligase RNF157

    PubMed Central

    Matz, A; Lee, S-J; Schwedhelm-Domeyer, N; Zanini, D; Holubowska, A; Kannan, M; Farnworth, M; Jahn, O; Göpfert, M C; Stegmüller, J

    2015-01-01

    Neuronal health is essential for the long-term integrity of the brain. In this study, we characterized the novel E3 ubiquitin ligase ring finger protein 157 (RNF157), which displays a brain-dominant expression in mouse. RNF157 is a homolog of the E3 ligase mahogunin ring finger-1, which has been previously implicated in spongiform neurodegeneration. We identified RNF157 as a regulator of survival in cultured neurons and established that the ligase activity of RNF157 is crucial for this process. We also uncovered that independently of its ligase activity, RNF157 regulates dendrite growth and maintenance. We further identified the adaptor protein APBB1 (amyloid beta precursor protein-binding, family B, member 1 or Fe65) as an interactor and proteolytic substrate of RNF157 in the control of neuronal survival. Here, the nuclear localization of Fe65 together with its interaction partner RNA-binding protein SART3 (squamous cell carcinoma antigen recognized by T cells 3 or Tip110) is crucial to trigger apoptosis. In summary, we described that the E3 ligase RNF157 regulates important aspects of neuronal development. PMID:25342469

  18. Regulation of neuronal survival and morphology by the E3 ubiquitin ligase RNF157.

    PubMed

    Matz, A; Lee, S-J; Schwedhelm-Domeyer, N; Zanini, D; Holubowska, A; Kannan, M; Farnworth, M; Jahn, O; Göpfert, M C; Stegmüller, J

    2015-04-01

    Neuronal health is essential for the long-term integrity of the brain. In this study, we characterized the novel E3 ubiquitin ligase ring finger protein 157 (RNF157), which displays a brain-dominant expression in mouse. RNF157 is a homolog of the E3 ligase mahogunin ring finger-1, which has been previously implicated in spongiform neurodegeneration. We identified RNF157 as a regulator of survival in cultured neurons and established that the ligase activity of RNF157 is crucial for this process. We also uncovered that independently of its ligase activity, RNF157 regulates dendrite growth and maintenance. We further identified the adaptor protein APBB1 (amyloid beta precursor protein-binding, family B, member 1 or Fe65) as an interactor and proteolytic substrate of RNF157 in the control of neuronal survival. Here, the nuclear localization of Fe65 together with its interaction partner RNA-binding protein SART3 (squamous cell carcinoma antigen recognized by T cells 3 or Tip110) is crucial to trigger apoptosis. In summary, we described that the E3 ligase RNF157 regulates important aspects of neuronal development. PMID:25342469

  19. Mutation screening of the ubiquitin ligase gene RNF135 in French patients with autism.

    PubMed

    Tastet, Julie; Decalonne, Loïc; Marouillat, Sylviane; Malvy, Joëlle; Thépault, Rose-Anne; Toutain, Annick; Paubel, Agathe; Tabagh, Refaat; Bénédetti, Hélène; Laumonnier, Frédéric; Barthélémy, Catherine; Bonnet-Brilhault, Frédérique; Andres, Christian R; Vourc'h, Patrick

    2015-12-01

    Many genes are now thought to confer susceptibility to autism. Despite the fact that this neuropsychiatric disease appears to be related to several different causes, common cellular and molecular pathways have emerged and point to synaptic dysfunction or cellular growth. Several studies have indicated the importance of the ubiquitin pathway in synaptic function and the aetiology of autism. Here, we focused on the ring finger protein 135 (RNF135) gene, encoding an E3 ubiquitin ligase expressed in the cortex and cerebellum, and located in the NF1 gene locus in 17q11.2, a region linked to autism. We carried out a genetic analysis of the coding sequence of RFN135 in a French cohort of patients with autism and observed a significantly increased frequency of genotypes carrying the rare allele of the rs111902263 (p.R115K) missense variant in patients (P=0.0019, odds ratio: 4.23, 95% confidence interval: 1.87-9.57). Particularly, three unrelated patients showed a homozygous genotype for K115, a situation not observed in the 1812 control individuals. Further cellular and molecular studies are required to elucidate the role of this gene and the variant K115 in brain development and neuronal function. PMID:26368817

  20. Ubiquitin ligase RNF167 regulates AMPA receptor-mediated synaptic transmission

    PubMed Central

    Lussier, Marc P.; Herring, Bruce E.; Nasu-Nishimura, Yukiko; Neutzner, Albert; Karbowski, Mariusz; Youle, Richard J.; Nicoll, Roger A.; Roche, Katherine W.

    2012-01-01

    AMPA receptors (AMPARs) mediate the majority of fast excitatory neurotransmission, and their density at postsynaptic sites determines synaptic strength. Ubiquitination is a posttranslational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167. Predominantly lysosomal, a subpopulation of RNF167 is located on the surface of cultured neurons. Using a RING mutant RNF167 or a specific shRNA to eliminate endogenous RNF167, we demonstrate that AMPAR surface expression increases in hippocampal neurons with disrupted RNF167 activity and that RNF167 is involved in activity-dependent ubiquitination of AMPARs. In addition, RNF167 regulates synaptic AMPAR currents, whereas synaptic NMDAR currents are unaffected. Therefore, our study identifies RNF167 as a selective regulator of AMPAR-mediated neurotransmission and expands our understanding of how ubiquitination dynamically regulates excitatory synapses. PMID:23129617

  1. Membrane-Associated Ubiquitin Ligase SAUL1 Suppresses Temperature- and Humidity-Dependent Autoimmunity in Arabidopsis.

    PubMed

    Disch, Eva-Maria; Tong, Meixuezi; Kotur, Tanja; Koch, Gerald; Wolf, Carl-Asmus; Li, Xin; Hoth, Stefan

    2016-01-01

    Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid-dependent and pathogenesis-related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, or Hyaloperonospora arabidopsidis Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell-wall depositions. Confocal analysis on aniline blue-stained leaf sections and cellular universal micro spectrophotometry further showed that these cell-wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4 but not SAG101. Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

  2. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  3. The ubiquitin E3 ligase TRAF6 exacerbates pathological cardiac hypertrophy via TAK1-dependent signalling

    PubMed Central

    Ji, Yan-Xiao; Zhang, Peng; Zhang, Xiao-Jing; Zhao, Yi-Chao; Deng, Ke-Qiong; Jiang, Xi; Wang, Pi-Xiao; Huang, Zan; Li, Hongliang

    2016-01-01

    Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a ubiquitin E3 ligase that regulates important biological processes. However, the role of TRAF6 in cardiac hypertrophy remains unknown. Here, we show that TRAF6 levels are increased in human and murine hypertrophied hearts, which is regulated by reactive oxygen species (ROS) production. Cardiac-specific Traf6 overexpression exacerbates cardiac hypertrophy in response to pressure overload or angiotensin II (Ang II) challenge, whereas Traf6 deficiency causes an alleviated hypertrophic phenotype in mice. Mechanistically, we show that ROS, generated during hypertrophic progression, triggers TRAF6 auto-ubiquitination that facilitates recruitment of TAB2 and its binding to transforming growth factor beta-activated kinase 1 (TAK1), which, in turn, enables the direct TRAF6–TAK1 interaction and promotes TAK1 ubiquitination. The binding of TRAF6 to TAK1 and the induction of TAK1 ubiquitination and activation are indispensable for TRAF6-regulated cardiac remodelling. Taken together, we define TRAF6 as an essential molecular switch leading to cardiac hypertrophy in a TAK1-dependent manner. PMID:27249171

  4. E3 ubiquitin ligase RNF13 involves spatial learning and assembly of the SNARE complex.

    PubMed

    Zhang, Qiang; Li, Yanfeng; Zhang, Lei; Yang, Nan; Meng, Jiao; Zuo, Pingping; Zhang, Yong; Chen, Jie; Wang, Li; Gao, Xiang; Zhu, Dahai

    2013-01-01

    Changes in the structure and number of synapses modulate learning, memory and cognitive disorders. Ubiquitin-mediated protein modification is a key mechanism for regulating synaptic activity, though the precise control of this process remains poorly understood. RING finger protein 13 (RNF13) is a recently identified E3 ubiquitin ligase, and its in vivo function remains completely unknown. We show here that genetic deletion of RNF13 in mice leads to a significant deficit in spatial learning as determined by the Morris water maze test and Y-maze learning test. At the ultrastructral level, the synaptic vesicle density was decreased and the area of the active zone was increased at hippocampal synapses of RNF13-null mice compared with those of wild-type littermates. We found no change in the levels of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex proteins in the hippocampus of RNF13-null mice, but impaired SNARE complex assembly. RNF13 directly interacted with snapin, a SNAP-25-interacting protein. Interestingly, snapin was ubiquitinated by RNF13 via the lysine-29 conjugated polyubiquitin chain, which in turn promoted the association of snapin with SNAP-25. Consistently, we found an attenuated interaction between snapin and SNAP-25 in the RNF13-null mice. Therefore, these results suggest that RNF13 is involved in the regulation of the SNARE complex, which thereby controls synaptic function.

  5. Force Dependent Biotinylation of Myosin IIA by α-Catenin Tagged with a Promiscuous Biotin Ligase

    PubMed Central

    Ueda, Shuji; Blee, Alexandra M.; Macway, Katherine G.; Renner, Derrick J.; Yamada, Soichiro

    2015-01-01

    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  6. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

    PubMed Central

    Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

    2016-01-01

    The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity. PMID:26964677

  7. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

    NASA Astrophysics Data System (ADS)

    Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

    2016-03-01

    The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

  8. The Ubiquitin Ligase SCF(Ucc1) Acts as a Metabolic Switch for the Glyoxylate Cycle.

    PubMed

    Nakatsukasa, Kunio; Nishimura, Takashi; Byrne, Stuart D; Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Okumura, Fumihiko; Kamura, Takumi

    2015-07-01

    Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis. Moreover, in vitro analysis demonstrates that oxaloacetate regenerated through the glyoxylate cycle induces a conformational change in citrate synthase and inhibits its recognition and ubiquitination by SCF(Ucc1), suggesting the existence of an oxaloacetate-dependent positive feedback loop that stabilizes citrate synthase. We propose that SCF(Ucc1)-mediated regulation of citrate synthase acts as a metabolic switch for the glyoxylate cycle in response to changes in carbon source, thereby ensuring metabolic versatility and flexibility.

  9. Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

    PubMed Central

    Wiedmann, M; Czajka, J; Barany, F; Batt, C A

    1992-01-01

    A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes. Images PMID:1482171

  10. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

    PubMed

    Nguyen, Giang K T; Wang, Shujing; Qiu, Yibo; Hemu, Xinya; Lian, Yilong; Tam, James P

    2014-09-01

    Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations. PMID:25038786

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

    PubMed Central

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

    2014-01-01

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

  12. Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.

    PubMed

    Franciosini, Anna; Serino, Giovanna

    2016-01-01

    CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure. PMID:27424742

  13. Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design

    PubMed Central

    Zhong, Shijun; Chen, Xi; Zhu, Xiao; Dziegielewska, Barbara; Bachman, Kurtis E.; Ellenberger, Tom; Ballin, Jeff D.; Wilson, Gerald M.; Tomkinson, Alan E.; MacKerell, Alexander D.

    2009-01-01

    Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies. PMID:18630893

  14. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

    SciTech Connect

    Meier, Christoph; Carter, Lester G.; Winter, Graeme; Owens, Ray J.; Stuart, David I.; Esnouf, Robert M.

    2007-03-01

    The structure of 5-formyltetrahydrofolate cyclo-ligase from B. anthracis determined by X-ray crystallography at a resolution of 1.6 Å is described. Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

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

    SciTech Connect

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

    2005-01-01

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

  16. Biochemical and structural characterization of DNA ligases from bacteria and archaea

    PubMed Central

    Pergolizzi, Giulia; Wagner, Gerd K.; Bowater, Richard P.

    2016-01-01

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β-NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesized novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. PMID:27582505

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

    PubMed Central

    Nielsen, Sofie V.; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus

    2016-01-01

    The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is

  18. Functional significance of glutamate-cysteine ligase modifier for erythrocyte survival in vitro and in vivo.

    PubMed

    Föller, M; Harris, I S; Elia, A; John, R; Lang, F; Kavanagh, T J; Mak, T W

    2013-10-01

    Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm(-/-)) mice. Erythrocytes from gclm(-/-) mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm(-/-) erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm(-/-) mice showed dramatically increased hemolysis compared with gclm(+/+) controls. In addition, PHZ-treated gclm(-/-) mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm(+/+) mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm(-/-) mice and none in gclm(+/+) controls. In fact, 24 h after treatment, kidney function began to diminish in gclm(-/-) mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm(+/+) mice survived, 90% of PHZ-treated gclm(-/-) mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm(-/-) erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm(+/+) erythrocytes, an effect at least partially due to increased cytosolic Ca(2+) concentration. Under resting conditions, gclm(-/-) mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.

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

    PubMed

    Lee, Kyounghee; Seo, Pil Joon

    2015-01-01

    Ethylene regulates a variety of physiological processes, such as flowering, senescence, abscission, and fruit ripening. In particular, leaf expansion is also controlled by ethylene in Arabidopsis. Exogenous treatment with ethylene inhibits leaf expansion, and consistently, ethylene insensitive mutants show increased leaf area. Here, we report that the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) regulates leaf expansion in an ethylene signaling pathway. The HOS1-deficient mutant showed reduced leaf area and was insensitive to ethylene perception inhibitor, silver thiosulfate (STS). Accordingly, genes encoding ethylene signaling components were significantly up-regulated in hos1-3. This study demonstrates that the HOS1 protein is involved in ethylene signal transduction for the proper regulation of leaf expansion possibly under environmentally stressful conditions. PMID:25848954

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

    PubMed Central

    Baehr, Leslie M.

    2014-01-01

    Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered. PMID:25096180

  1. NADPH oxidases promote apoptosis by activating ZNRF1 ubiquitin ligase in neurons treated with an exogenously applied oxidant

    PubMed Central

    Wakatsuki, Shuji; Araki, Toshiyuki

    2016-01-01

    ABSTRACT Reactive oxygen species (ROS) play an important role in causing neuronal death in a number of neurological disorders. We recently reported that ROS serve as a signal to activate neuronal apoptosis and axonal degeneration by activating ZNRF1 (zinc- and RING-finger 1), a ubiquitin ligase that targets AKT for proteasomal degradation in neurons. In the present study, we showed that the NADPH oxidase family of molecules is required for ZNRF1 activation by epidermal growth factor receptor (EGFR)-dependent phosphorylation in response to axonal injury. We herein demonstrate that NADPH oxidases promote apoptosis by activating ZNRF1, even in neurons treated with an exogenously applied oxidant. These results suggest an important role for NADPH oxidase in the initiation/promotion of neuronal degeneration by increasing ROS in close proximity to protein machineries, including those for ZNRF1 and EGFR, thereby promoting neuronal degeneration. PMID:27195063

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

    PubMed

    Lee, Kyounghee; Seo, Pil Joon

    2015-01-01

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

  3. Histone Modification of Nedd4 Ubiquitin Ligase Controls the Loss of AMPA Receptors and Cognitive Impairment Induced by Repeated Stress

    PubMed Central

    Wei, Jing; Xiong, Zhe; Lee, Janine B.; Cheng, Jia; Duffney, Lara J.; Matas, Emmanuel

    2016-01-01

    Stress and the major stress hormone corticosterone induce profound influences in the brain. Altered histone modification and transcriptional dysfunction have been implicated in stress-related mental disorders. We previously found that repeated stress caused an impairment of prefrontal cortex (PFC)-mediated cognitive functions by increasing the ubiquitination and degradation of AMPA-type glutamate receptors via a mechanism depending on the E3 ubiquitin ligase Nedd4. Here, we demonstrated that in PFC of repeatedly stressed rats, active glucocorticoid receptor had the increased binding to the glucocorticoid response element of histone deacetylase 2 (HDAC2) promoter, resulting in the upregulation of HDAC2. Inhibition or knock-down of HDAC2 blocked the stress-induced impairment of synaptic transmission, AMPAR expression, and recognition memory. Furthermore, we found that, in stressed animals, the HDAC2-dependent downregulation of histone methyltransferase Ehmt2 (G9a) led to the loss of repressive histone methylation at the Nedd4-1 promoter and the transcriptional activation of Nedd4. These results have provided an epigenetic mechanism and a potential treatment strategy for the detrimental effects of chronic stress. SIGNIFICANCE STATEMENT Prolonged stress exposure can induce altered histone modification and transcriptional dysfunction, which may underlie the profound influence of stress in regulating brain functions. We report an important finding about the epigenetic mechanism controlling the detrimental effects of repeated stress on synaptic transmission and cognitive function. First, it has revealed the stress-induced alteration of key epigenetic regulators HDAC2 and Ehmt2, which determines the synaptic and behavioral effects of repeated stress. Second, it has uncovered the stress-induced histone modification of the target gene Nedd4, an E3 ligase that is critically involved in the ubiquitination and degradation of AMPA receptors and cognition. Third, it has provided

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

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

    2011-12-01

    Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization. PMID:22000681

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

    PubMed

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

    2016-08-08

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

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

    PubMed Central

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

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  9. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Guzmán, Plinio

    2012-01-01

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

  12. SILENCING OF 4-COUMARATE-CoA LIGASE IN PINUS RADIATA, A CONIFEROUS GYMNOSPERM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme 4-coumarate-CoA ligase (4CL) is involved in the general phenylpropanoid pathway and provides monolignol precursors such as p-coumaroyl-CoA, ultimately for the biosynthesis of lignin. Recombinant studies designed to assess the role of 4CL in the lignification process have focused on angios...

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

    PubMed

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

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  14. Another tier for caspase regulation: IAPs as NEDD8 E3 ligases.

    PubMed

    Benjamin, Sigi; Steller, Hermann

    2010-12-14

    Many inhibitor of apoptosis proteins (IAPs) function as E3 ligases to ubiquitinate important cell death proteins, including caspases. Broemer et al. (2010) report recently in Molecular Cell that IAPs can also inhibit caspases by promoting conjugation of the UBL NEDD8.

  15. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

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

    PubMed

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

    2011-04-22

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

  17. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  18. Identification of dynamical hinge points of the L1 ligase molecular switch.

    PubMed

    Giambasu, George M; Lee, Tai-Sung; Sosa, Carlos P; Robertson, Michael P; Scott, William G; York, Darrin M

    2010-04-01

    The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5' to 3' phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive-active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.

  19. Molecular dynamics reveal the essential role of linker motions in the function of cullin-RING E3 ligases

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2010-01-01

    Tagging proteins by polyubiquitin is a key step in protein degradation. Cullin-RING E3 ubiquitin ligases (CRLs) facilitate ubiquitin transfer from the E2 conjugating enzyme to the substrate; yet, crystallography indicates a large distance between the E2 and the substrate, raising the question of how this distance is bridged in the ubiquitin transfer reaction. Here, we demonstrate that the linker motions in the substrate binding proteins can allosterically shorten this distance to facilitate this crucial ubiquitin transfer step, and increase this distance to allow polyubiquitination. We performed molecular dynamics simulations for five substrate binding proteins, Skp2, Fbw7, β-TrCP1, Cdc4, and pVHL, in two forms: bound to their substrates, and bound to both substrate and adaptor. The adaptor connects the substrate binding proteins to the cullin. In the bound-to-both forms of all cases, we observed rotations of the substrate binding domain, shortening the gap between the tip of the substrate peptide and the E2 active site by 7~12Å compared to the crystal structures. Overall, together with our earlier simulations of the unbound and the bound-to-adaptor forms, the emerging picture is that the maximum 51~73Å distance between the substrate binding domain and the E2 active site in the modeled unbound forms of these five proteins shrinks to a minimum of 39~49Å in the bound-to-both forms. This large distance range, the result of allosterically-controlled linker motions, facilitates the ubiquitin transfer and polyubiquitination, and as such argues that the cullin-RING E3 ubiquitin ligase is under conformational control. We further observed that substrate binding proteins with multiple substrate acceptor lysines have larger distance range between the substrate and the E2 as compared to β-TrCP1, with only one acceptor lysine. PMID:20083119

  20. Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis.

    PubMed

    Remus, Barbara S; Schwer, Beate; Shuman, Stewart

    2016-10-01

    Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2',3'-cyclic-PO4 and 5'-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.

  1. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-18

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

  5. Mechanistic assessment of DNA ligase as an antibacterial target in Staphylococcus aureus.

    PubMed

    Podos, Steven D; Thanassi, Jane A; Pucci, Michael J

    2012-08-01

    We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD(+)-dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [K(i)] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC(50)] = 28 nM) or its isolated adenylation domain (IC(50) = 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistant S. aureus (MSSA and MRSA) strains (MIC = 1.0 μg/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 × 10(-7)) of high-level resistant mutants (MIC > 64) with associated ligA lesions. There were no observable effects on growth rate in these mutants. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains. In vitro characterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrate K(m) and a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy. PMID:22585221

  6. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway. PMID:23677922

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

    PubMed

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

    2016-01-28

    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

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

    PubMed Central

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

    2015-01-01

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

  9. Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling

    PubMed Central

    Joo, Hyunhee; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling. PMID:27439598

  10. Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling.

    PubMed

    Joo, Hyunhee; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling. PMID:27439598

  11. SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

    PubMed Central

    Ohkuni, Kentaro; Takahashi, Yoshimitsu; Fulp, Alyona; Lawrimore, Josh; Au, Wei-Chun; Pasupala, Nagesh; Levy-Myers, Reuben; Warren, Jack; Strunnikov, Alexander; Baker, Richard E.; Kerscher, Oliver; Bloom, Kerry; Basrai, Munira A.

    2016-01-01

    Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast and flies and tumorigenesis in human cells; thus defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast shows that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1, since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization than either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability. PMID:26960795

  12. Non-thermal plasma induces AKT degradation through turn-on the MUL1 E3 ligase in head and neck cancer

    PubMed Central

    Kim, Sun-Yong; Kim, Haeng-Jun; Kang, Sung Un; Kim, Yang Eun; Park, Ju Kyeong; Shin, Yoo Seob; Kim, Yeon Soo; Lee, Keunho; Kim, Chul-Ho

    2015-01-01

    Recent research on non-thermal plasma (NTP, an ionized gas) has identified it as a novel cancer therapeutic tool. However, the molecular mechanism remains unclear. In this study, we demonstrated NTP induced cell death of head and neck cancer (HNC) through the AKT ubiquitin–proteasome system. NTP increased the gene expression of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), an E3 ligase for AKT, and NTP-induced HNC cell death was prevented by MUL1 siRNA. We also showed that MUL1 inhibited the level of AKT and p-AKT and MUL1 expression was increased by NTP-induced ROS. Furthermore, we optimized and manufactured a new type of NTP, a liquid type of NTP (LTP). In syngeneic and xenograft in vivo tumor models, LTP inhibited tumor progression by increasing the MUL1 level and reducing p-AKT levels, indicating that LTP also has an anti-cancer effect through the same mechanism as that of NTP. Taken together, our results suggest that NTP and LTP have great potential for HNC therapy. PMID:26450902

  13. “Ubiquitylation: mechanism and functions“ Review series: RBR E3-ligases at work

    PubMed Central

    Smit, Judith J; Sixma, Titia K

    2014-01-01

    The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region. PMID:24469331

  14. AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism.

    PubMed Central

    Montecucco, A; Ciarrocchi, G

    1988-01-01

    In the presence of AMP and Mg2+, a covalently closed duplex DNA containing negative superhelical turns was treated with DNA ligase isolated from bacteriophage T4-infected E. coli. This resulted in the gradual and not sudden loss of superhelical turns as for example in the case of type I DNA topoisomerase. All DNA products remain covalently closed. Since T4 enzyme-mediated DNA relaxation is inhibited by both pyrophosphate and by ATP this suggests that DNA relaxing and DNA joining activities probably coincide. EDTA addition in the presence of a large excess of enzyme, induces the formation of nicked DNA products while protein denaturing treatments are not very effective. Our observations might suggest an involvement of the relaxing activity of DNA ligase during the ligation process. Images PMID:3137526

  15. The interplay between DSL proteins and ubiquitin ligases in Notch signaling.

    PubMed

    Pitsouli, Chrysoula; Delidakis, Christos

    2005-09-01

    Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. We now show that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. We also characterize a mutation in mib1, a Drosophila homolog of mind bomb, another Delta-targeting ubiquitin ligase from zebrafish. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling.

  16. Inhibitor of apoptosis proteins as E3 ligases for ubiquitin and NEDD8.

    PubMed

    Kamada, Shinji

    2013-04-01

    The inhibitors of apoptosis proteins (IAPs) are endogenous inhibitors for apoptosis. Apoptosis is carried out by caspases, which are the family of cystein proteases. IAPs regulate caspases through two conserved regions, the baculovirus IAP repeats (BIRs) and the really interesting new gene (RING) domains. Although the BIRs are responsible for binding to caspases, the RING domain can act as a ubiquitin-E3 ligase, leading to ubiquitylation of IAPs themselves and their pro-apoptotic IAP counterparts such as caspases. Recently, it is reported that another ubiquitin-like protein, neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8), is also involved in the regulation of apoptosis through neddylation of caspases mediated by IAPs. On the contrary, the results against the function of IAPs as a NEDD8-E3 ligase are also suggested. This review presents the summary of IAPs, caspases, and the ubiquitin-proteasome system and how their interactions influence the regulation of apoptosis.

  17. IAPs as E3 ligases of Rac1: shaping the move.

    PubMed

    Oberoi-Khanuja, Tripat Kaur; Rajalingam, Krishnaraj

    2012-01-01

    Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling.

  18. Structurally complex and highly active RNA ligases derived from random RNA sequences

    NASA Technical Reports Server (NTRS)

    Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

    1995-01-01

    Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

  19. An improved smaller biotin ligase for BioID proximity labeling

    PubMed Central

    Kim, Dae In; Jensen, Samuel C.; Noble, Kyle A.; KC, Birendra; Roux, Kenneth H.; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    The BioID method uses a promiscuous biotin ligase to detect protein–protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein–protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. PMID:26912792

  20. Patched-1 proapoptotic activity is downregulated by modification of K1413 by the E3 ubiquitin-protein ligase Itchy homolog.

    PubMed

    Chen, Xiaole L; Chinchilla, Pilar; Fombonne, Joanna; Ho, Lan; Guix, Catherine; Keen, James H; Mehlen, Patrick; Riobo, Natalia A

    2014-10-01

    The Hedgehog (Hh) receptor Patched-1 (PTCH1) opposes the activation of Gli transcription factors and induces cell death through a Gli-independent pathway. Here, we report that the C-terminal domain (CTD) of PTCH1 interacts with and is ubiquitylated on K1413 by the E3 ubiquitin-protein ligase Itchy homolog (Itch), a Nedd4 family member. Itch induces the ubiquitylation of K1413, the reduction of PTCH1 levels at the plasma membrane, and degradation, activating Gli transcriptional activity in the absence of Hh ligands. Silencing of Itch stabilizes PTCH1 and increases its level of retention at the plasma membrane. Itch is the preferential PTCH1 E3 ligase in the absence of Hh ligands, since of the other seven Nedd4 family members, only WW domain-containing protein 2 (WWP2) showed a minor redundant role. Like Itch depletion, mutation of the ubiquitylation site (K1314R) resulted in the accumulation of PTCH1 at the plasma membrane, prolongation of its half-life, and increased cell death by hyperactivation of caspase-9. Remarkably, Itch is the main determinant of PTCH1 stability under resting conditions but not in response to Sonic Hedgehog. In conclusion, our findings reveal that Itch is a key regulator of ligand-independent Gli activation and noncanonical Hh signaling by the governance of basal PTCH1 internalization and degradation. PMID:25092867

  1. The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

    PubMed

    Li, Ying; Zhang, Liang; Li, Dekuan; Liu, Zhibin; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2016-03-01

    The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability. PMID:26386272

  2. Patched-1 Proapoptotic Activity Is Downregulated by Modification of K1413 by the E3 Ubiquitin-Protein Ligase Itchy Homolog

    PubMed Central

    Chen, Xiaole L.; Chinchilla, Pilar; Fombonne, Joanna; Ho, Lan; Guix, Catherine; Keen, James H.; Mehlen, Patrick

    2014-01-01

    The Hedgehog (Hh) receptor Patched-1 (PTCH1) opposes the activation of Gli transcription factors and induces cell death through a Gli-independent pathway. Here, we report that the C-terminal domain (CTD) of PTCH1 interacts with and is ubiquitylated on K1413 by the E3 ubiquitin-protein ligase Itchy homolog (Itch), a Nedd4 family member. Itch induces the ubiquitylation of K1413, the reduction of PTCH1 levels at the plasma membrane, and degradation, activating Gli transcriptional activity in the absence of Hh ligands. Silencing of Itch stabilizes PTCH1 and increases its level of retention at the plasma membrane. Itch is the preferential PTCH1 E3 ligase in the absence of Hh ligands, since of the other seven Nedd4 family members, only WW domain-containing protein 2 (WWP2) showed a minor redundant role. Like Itch depletion, mutation of the ubiquitylation site (K1314R) resulted in the accumulation of PTCH1 at the plasma membrane, prolongation of its half-life, and increased cell death by hyperactivation of caspase-9. Remarkably, Itch is the main determinant of PTCH1 stability under resting conditions but not in response to Sonic Hedgehog. In conclusion, our findings reveal that Itch is a key regulator of ligand-independent Gli activation and noncanonical Hh signaling by the governance of basal PTCH1 internalization and degradation. PMID:25092867

  3. Nuclear localization of glutamate-cysteine ligase is associated with proliferation in head and neck squamous cell carcinoma

    PubMed Central

    DEQUANTER, DIDIER; VAN DE VELDE, MAUREEN; BAR, ISABELLE; NUYENS, VINCENT; ROUSSEAU, ALEXANDRE; NAGY, NATHALIE; VANHAMME, LUC; VANHAEVERBEEK, MICHEL; BROHÉE, DANY; DELRÉE, PAUL; BOUDJELTIA, KARIM; LOTHAIRE, PHILIPPE; UZUREAU, PIERRICK

    2016-01-01

    Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation. PMID:27284370

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

    PubMed

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

    2015-08-15

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

  5. Enzyme–adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface

    PubMed Central

    Williamson, Adele; Rothweiler, Ulli; Schrøder Leiros, Hanna-Kirsti

    2014-01-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme–adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date. PMID:25372693

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

    PubMed Central

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

    2014-01-01

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

  7. Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

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

    SciTech Connect

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

    2009-06-04

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

  9. Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata.

    PubMed

    Sinha, Krishna Murari; Hines, Jane C; Ray, Dan S

    2006-01-01

    The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

  10. Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

    PubMed Central

    Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

    2015-01-01

    E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. ZNF451 catalytic module contains tandem SUMO interaction motifs (SIMs) bridged by a Proline-Leucine-Arginine-Proline (PLRP) motif. The first SIM and PLRP motif engage thioester charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the backside of E2. We show that ZNF451 is SUMO2 specific and that SUMO-modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase. PMID:26524494

  11. E3 Ubiquitin Ligases Pellinos as Regulators of Pattern Recognition Receptor Signaling and Immune responses

    PubMed Central

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

    2015-01-01

    SUMMARY Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an IL-1 receptor-associated kinase homologue in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2 and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g., Pellino-1 being a negative regulator in T-lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we have summarized current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and TNF receptors, and discuss Pellino’s role in sepsis and infectious diseases, as well as in autoimmune, inflammatory and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies. PMID:26085210

  12. Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers[W

    PubMed Central

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A.; Widhalm, Joshua R.; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M.; Cooper, Bruce R.; D’Auria, John C.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-01-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. PMID:22649270

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

    PubMed Central

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

    2015-01-01

    Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1–3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state. PMID:26701932

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  16. SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination

    PubMed Central

    Han, Jinhua; Liu, Ting; Huen, Michael S.Y.; Hu, Lin; Chen, Zhiqiu

    2014-01-01

    Translesion DNA synthesis (TLS) is a universal DNA damage tolerance mechanism conserved from yeast to mammals. A key event in the regulation of TLS is the monoubiquitination of proliferating cell nuclear antigen (PCNA). Extensive evidence indicates that the RAD6–RAD18 ubiquitin-conjugating/ligase complex specifically monoubiquitinates PCNA and regulates TLS repair. However, the mechanism by which the RAD6–RAD18 complex is targeted to PCNA has remained elusive. In this study, we used an affinity purification approach to isolate the PCNA-containing complex and have identified SIVA1 as a critical regulator of PCNA monoubiquitination. We show that SIVA1 constitutively interacts with PCNA via a highly conserved PCNA-interacting peptide motif. Knockdown of SIVA1 compromised RAD18-dependent PCNA monoubiquitination and Polη focus formation, leading to elevated ultraviolet sensitivity and mutation. Furthermore, we demonstrate that SIVA1 interacts with RAD18 and serves as a molecular bridge between RAD18 and PCNA, thus targeting the E3 ligase activity of RAD18 onto PCNA. Collectively, our results provide evidence that the RAD18 E3 ligase requires an accessory protein for binding to its substrate PCNA. PMID:24958773

  17. Solving the SAT problem using a DNA computing algorithm based on ligase chain reaction.

    PubMed

    Wang, Xiaolong; Bao, Zhenmin; Hu, Jingjie; Wang, Shi; Zhan, Aibin

    2008-01-01

    A new DNA computing algorithm based on a ligase chain reaction is demonstrated to solve an SAT problem. The proposed DNA algorithm can solve an n-variable m-clause SAT problem in m steps and the computation time required is O (3m+n). Instead of generating the full-solution DNA library, we start with an empty test tube and then generate solutions that partially satisfy the SAT formula. These partial solutions are then extended step by step by the ligation of new variables using Taq DNA ligase. Correct strands are amplified and false strands are pruned by a ligase chain reaction (LCR) as soon as they fail to satisfy the conditions. If we score and sort the clauses, we can use this algorithm to markedly reduce the number of DNA strands required throughout the computing process. In a computer simulation, the maximum number of DNA strands required was 2(0.48n) when n=50, and the exponent ratio varied inversely with the number of variables n and the clause/variable ratio m/n. This algorithm is highly space-efficient and error-tolerant compared to conventional brute-force searching, and thus can be scaled-up to solve large and hard SAT problems. PMID:17904730

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

    PubMed

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

    2015-07-01

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

  19. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  20. Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis.

    PubMed

    Jeon, Hyo Jeong; Shin, Hea-Jin; Choi, Jeong Jin; Hoe, Hyang-Sook; Kim, Hyun-Kyu; Suh, Se Won; Kwon, Suk-Tae

    2004-08-01

    The crystal structure of NAD+-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wild-type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type. PMID:15268945

  1. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    PubMed Central

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

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

    PubMed Central

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

    2016-01-01

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

  3. Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

    PubMed Central

    Cheng, Qian; Song, Tanjing; Chen, Lihong

    2014-01-01

    The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels. PMID:24842904

  4. Coevolution of RtcB and Archease created a multiple-turnover RNA ligase.

    PubMed

    Desai, Kevin K; Beltrame, Amanda L; Raines, Ronald T

    2015-11-01

    RtcB is a noncanonical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

  5. Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2).

    PubMed

    Pan, I-Chun; Tsai, Huei-Hsuan; Cheng, Ya-Tan; Wen, Tuan-Nan; Buckhout, Thomas J; Schmidt, Wolfgang

    2015-10-01

    Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)(1) and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126.

  6. Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2)*

    PubMed Central

    Pan, I-Chun; Tsai, Huei-Hsuan; Cheng, Ya-Tan; Wen, Tuan-Nan; Buckhout, Thomas J.; Schmidt, Wolfgang

    2015-01-01

    Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)1 and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126. PMID:26253232

  7. A Non-Canonical Function of Gβ as a Subunit of E3 Ligase in Targeting GRK2 Ubiquitylation.

    PubMed

    Zha, Zhengyu; Han, Xiaoran; Smith, Matthew D; Liu, Yang; Giguère, Patrick M; Kopanja, Dragana; Raychaudhuri, Pradip; Siderovski, David P; Guan, Kun-Liang; Lei, Qun-Ying; Xiong, Yue

    2015-06-01

    G protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors, regulate a wide range of physiological processes, and are the major targets of pharmaceutical drugs. Canonical signaling from GPCRs is relayed to intracellular effector proteins by trimeric G proteins, composed of α, β, and γ subunits (Gαβγ). Here, we report that G protein β subunits (Gβ) bind to DDB1 and that Gβ2 targets GRK2 for ubiquitylation by the DDB1-CUL4A-ROC1 ubiquitin ligase. Activation of GPCR results in PKA-mediated phosphorylation of DDB1 at Ser645 and its dissociation from Gβ2, leading to increase of GRK2 protein. Deletion of Cul4a results in cardiac hypertrophy in male mice that can be partially rescued by the deletion of one Grk2 allele. These results reveal a non-canonical function of the Gβ protein as a ubiquitin ligase component and a mechanism of feedback regulation of GPCR signaling.

  8. Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas

    PubMed Central

    Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B.; Zhang, Donna D.; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

    2016-01-01

    Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity. PMID:27573825

  9. Endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls B-cell immunity through degradation of the death receptor CD95/Fas.

    PubMed

    Kong, Sinyi; Yang, Yi; Xu, Yuanming; Wang, Yajun; Zhang, Yusi; Melo-Cardenas, Johanna; Xu, Xiangping; Gao, Beixue; Thorp, Edward B; Zhang, Donna D; Zhang, Bin; Song, Jianxun; Zhang, Kezhong; Zhang, Jianning; Zhang, Jinping; Li, Huabin; Fang, Deyu

    2016-09-13

    Humoral immunity involves multiple checkpoints during B-cell development, maturation, and activation. The cell death receptor CD95/Fas-mediated apoptosis plays a critical role in eliminating the unwanted activation of B cells by self-reactive antigens and in maintaining B-cell homeostasis through activation-induced B-cell death (AICD). The molecular mechanisms controlling AICD remain largely undefined. Herein, we show that the E3 ubiquitin ligase Hrd1 protected B cells from activation-induced cell death by degrading the death receptor Fas. Hrd1-null B cells exhibited high Fas expression during activation and rapidly underwent Fas-mediated apoptosis, which could be largely inhibited by FasL neutralization. Fas mutation in Hrd1 KO mice abrogated the increase in B-cell AICD. We identified Hrd1 as the first E3 ubiquitin ligase of the death receptor Fas and Hrd1-mediated Fas destruction as a molecular mechanism in regulating B-cell immunity. PMID:27573825

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2016-07-01

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

  12. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  13. Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase

    PubMed Central

    Franklin, Christopher C.; Backos, Donald S.; Mohar, Isaac; White, Collin C.; Forman, Henry J.; Kavanagh, Terrance J.

    2009-01-01

    Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as γ-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique γ-carboxyl linkage from glutamate to cysteine and requires ATP and Mg++ as cofactors in this reaction. GCLM increases the Vmax and Kcat of GCLC, decreases the Km for glutamate and ATP, and increases the Ki for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans. PMID:18812186

  14. Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

    SciTech Connect

    Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M.

    2012-10-24

    To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    2009-01-01

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

  17. Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

    SciTech Connect

    Stanley,B.; Ehrlich, E.; Short, L.; Yu, Y.; Xiao, Z.; Yu, X.; Xiong, Y.

    2008-01-01

    Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

  18. Mitochondrial E3 ligase March5 maintains stemness of mouse ES cells via suppression of ERK signalling.

    PubMed

    Gu, Hao; Li, Qidong; Huang, Shan; Lu, Weiguang; Cheng, Fangyuan; Gao, Ping; Wang, Chen; Miao, Lin; Mei, Yide; Wu, Mian

    2015-01-01

    Embryonic stem cells (ESCs) possess pluripotency, which is the capacity of cells to differentiate into all lineages of the mature organism. Increasing evidence suggests that the pluripotent state of ESCs is regulated by a combination of extrinsic and intrinsic factors. The underlying mechanisms, however, are not completely understood. Here, we show that March5, an E3 ubiquitin ligase, is involved in maintaining mouse-ESC (mESC) pluripotency. Knockdown of March5 in mESCs led to differentiation from naive pluripotency. Mechanistically, as a transcriptional target of Klf4, March5 catalyses K63-linked polyubiquitination of Prkar1a, a negative regulatory subunit of PKA, to activate PKA, thereby inhibiting the Raf/MEK/ERK pathway. Moreover, March5 is able to replace a MEK/ERK inhibitor to maintain mESC pluripotency under serum-free culture conditions. In addition, March5 can partially replace the use of Klf4 for somatic cell reprogramming. Collectively, our study uncovers a role for the Klf4-March5-PKA-ERK pathway in maintaining the stemness properties of mESCs. PMID:26033541

  19. A genomic survey of HECT ubiquitin ligases in eukaryotes reveals independent expansions of the HECT system in several lineages.

    PubMed

    Grau-Bové, Xavier; Sebé-Pedrós, Arnau; Ruiz-Trillo, Iñaki

    2013-01-01

    The posttranslational modification of proteins by the ubiquitination pathway is an important regulatory mechanism in eukaryotes. To date, however, studies on the evolutionary history of the proteins involved in this pathway have been restricted to E1 and E2 enzymes, whereas E3 studies have been focused mainly in metazoans and plants. To have a wider perspective, here we perform a genomic survey of the HECT family of E3 ubiquitin-protein ligases, an important part of this posttranslational pathway, in genomes from representatives of all major eukaryotic lineages. We classify eukaryotic HECTs and reconstruct, by phylogenetic analysis, the putative repertoire of these proteins in the last eukaryotic common ancestor (LECA). Furthermore, we analyze the diversity and complexity of protein domain architectures of HECTs along the different extant eukaryotic lineages. Our data show that LECA had six different HECTs and that protein expansion and N-terminal domain diversification shaped HECT evolution. Our data reveal that the genomes of animals and unicellular holozoans considerably increased the molecular and functional diversity of their HECT system compared with other eukaryotes. Other eukaryotes, such as the Apusozoa Thecanomas trahens or the Heterokonta Phytophthora infestans, independently expanded their HECT repertoire. In contrast, plant, excavate, rhodophyte, chlorophyte, and fungal genomes have a more limited enzymatic repertoire. Our genomic survey and phylogenetic analysis clarifies the origin and evolution of different HECT families among eukaryotes and provides a useful phylogenetic framework for future evolutionary studies of this regulatory pathway.

  20. Human stanniocalcin-1 interacts with nuclear and cytoplasmic proteins and acts as a SUMO E3 ligase.

    PubMed

    dos Santos, Marcos Tadeu; Trindade, Daniel Maragno; Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Anastassopoulos, Filipe; Yunes, José Andres; Kobarg, Jörg

    2011-01-01

    Human stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in different physiological process, including angiogenesis, apoptosis and carcinogenesis. Here we identified STC1 as a putative molecular marker for the leukemic bone marrow microenvironment and identified new interacting protein partners for STC1. Seven selected interactions retrieved from yeast two-hybrid screens were confirmed by GST-pull down assays in vitro. The N-terminal region was mapped to be the region that mediates the interaction with cytoplasmic, mitochondrial and nuclear proteins. STC1 interacts with SUMO-1 and several proteins that have been shown to be SUMOylated and localized to SUMOylation related nuclear bodies. Although STC1 interacts with SUMO-1 and has a high theoretical prediction score for a SUMOylation site, endogenous co-immunoprecipitation and in vitro SUMOylation assays with the purified recombinant protein could not detect STC1 SUMOylation. However, when we tested STC1 for SUMO E3 ligase activity, we found in an in vitro assay, that it significantly increases the SUMOylation of two other proteins. Confocal microscopic subcellular localization studies using both transfected cells and specific antibodies for endogenous STC1 revealed a cytoplasmic and nuclear deposition, the latter in the form of some specific dot-like substructure resembling SUMOylation related nuclear bodies. Together, these findings suggest a new role for STC1 in SUMOylation pathways, in nuclear bodies.

  1. Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

    PubMed

    Fiesel, Fabienne C; Caulfield, Thomas R; Moussaud-Lamodière, Elisabeth L; Ogaki, Kotaro; Dourado, Daniel F A R; Flores, Samuel C; Ross, Owen A; Springer, Wolfdieter

    2015-08-01

    Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

  2. Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry.

    PubMed

    Su, Wen-Chi; Chen, Yung-Chia; Tseng, Chung-Hsin; Hsu, Paul Wei-Che; Tung, Kuo-Feng; Jeng, King-Song; Lai, Michael M C

    2013-10-22

    Influenza viruses, like other viruses, rely on host factors to support their life cycle as viral proteins usually "hijack," or collaborate with, cellular proteins to execute their functions. Identification and understanding of these factors can increase the knowledge of molecular mechanisms manipulated by the viruses and facilitate development of antiviral drugs. To this end, we developed a unique genome-wide pooled shRNA screen to search for cellular factors important for influenza A virus (IAV) replication. We identified an E3 ubiquitin ligase, Itch, as an essential factor for an early step in the viral life cycle. In Itch knockdown cells, the incorporation of viral ribonucleoprotein complex into endosomes was normal, but its subsequent release from endosomes and transport to the nucleus was retarded. In addition, upon virus infection, Itch was phosphorylated and recruited to the endosomes, where virus particles were located. Furthermore, Itch interacted with viral M1 protein and ubiquitinated M1 protein. Collectively, our findings unravel a critical role of Itch in mediating IAV release from the endosome and offer insights into the mechanism for IAV uncoating during virus entry. These findings also highlight the feasibility of pooled RNAi screening for exploring the cellular cofactors of lytic viruses. PMID:24101521

  3. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

  4. The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells.

    PubMed

    Fan, Lingling; Peng, Guihong; Hussain, Arif; Fazli, Ladan; Guns, Emma; Gleave, Martin; Qi, Jianfei

    2015-08-21

    Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

  5. SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis.

    PubMed

    Jia, Fengjuan; Wang, Chunyan; Huang, Jinguang; Yang, Guodong; Wu, Changai; Zheng, Chengchao

    2015-08-01

    Skp1-Cullin-F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na(+) homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na(+) accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis.

  6. The E3 ubiquitin ligase WWP1 regulates {Delta}Np63-dependent transcription through Lys63 linkages

    SciTech Connect

    Peschiaroli, Angelo; Scialpi, Flavia; Bernassola, Francesca; Sherbini, El Said El; Melino, Gerry

    2010-11-12

    Research highlights: {yields} WWP1 ubiquitylates {Delta}Np63 through conjugation of Lys63-linked poly-ubiquitin chains. {yields} WWP1 does not control {Delta}Np63 protein stability. {yields} WWP1 regulates {Delta}Np63-dependent transcription. -- Abstract: The transcription factor p63, a member of the p53 family, plays a crucial role in epithelial development and tumorigenesis through the regulation of epithelial progenitor cell proliferation, differentiation and apoptosis. Similarly to p53, p63 activity is regulated by post-translational modifications, including ubiquitylation. Here, we report that the WWP1 E3 ubiquitin ligase binds specifically to {Delta}Np63 isoform but it does not trigger {Delta}Np63 proteasome-dependent degradation. Accordingly, we found that WWP1-dependent ubiquitylation of {Delta}Np63 occurs through the formation of Lys63-linked poly-ubiquitin chains. Importantly, we found that WWP1 is able to increase {Delta}Np63-dependent transcription and depletion of WWP1 in human primary keratinocytes induces cell cycle arrest. All together these results indicate that WWP1 regulates {Delta}Np63 transcriptional activity, acting thus as a potential regulator of the proliferation and survival of epithelial-derived cells.

  7. Synaptic E3 Ligase SCRAPPER in Contextual Fear Conditioning: Extensive Behavioral Phenotyping of Scrapper Heterozygote and Overexpressing Mutant Mice

    PubMed Central

    Yao, Ikuko; Takao, Keizo; Miyakawa, Tsuyoshi; Ito, Seiji; Setou, Mitsutoshi

    2011-01-01

    SCRAPPER, an F-box protein coded by FBXL20, is a subunit of SCF type E3 ubiquitin ligase. SCRAPPER localizes synapses and directly binds to Rab3-interacting molecule 1 (RIM1), an essential factor for synaptic vesicle release, thus it regulates neural transmission via RIM1 degradation. A defect in SCRAPPER leads to neurotransmission abnormalities, which could subsequently result in neurodegenerative phenotypes. Because it is likely that the alteration of neural transmission in Scrapper mutant mice affect their systemic condition, we have analyzed the behavioral phenotypes of mice with decreased or increased the amount of SCRAPPER. We carried out a series of behavioral test batteries for Scrapper mutant mice. Scrapper transgenic mice overexpressing SCRAPPER in the hippocampus did not show any significant difference in every test argued in this manuscript by comparison with wild-type mice. On the other hand, heterozygotes of Scrapper knockout [SCR (+/−)] mice showed significant difference in the contextual but not cued fear conditioning test. In addition, SCR (+/−) mice altered in some tests reflecting anxiety, which implies the loss of functions of SCRAPPER in the hippocampus. The behavioral phenotypes of Scrapper mutant mice suggest that molecular degradation conferred by SCRAPPER play important roles in hippocampal-dependent fear memory formation. PMID:21390313

  8. Mitochondrial E3 ligase March5 maintains stemness of mouse ES cells via suppression of ERK signalling

    PubMed Central

    Gu, Hao; Li, Qidong; Huang, Shan; Lu, Weiguang; Cheng, Fangyuan; Gao, Ping; Miao, Lin; Mei, Yide; Wu, Mian

    2015-01-01

    Embryonic stem cells (ESCs) possess pluripotency, which is the capacity of cells to differentiate into all lineages of the mature organism. Increasing evidence suggests that the pluripotent state of ESCs is regulated by a combination of extrinsic and intrinsic factors. The underlying mechanisms, however, are not completely understood. Here, we show that March5, an E3 ubiquitin ligase, is involved in maintaining mouse-ESC (mESC) pluripotency. Knockdown of March5 in mESCs led to differentiation from naive pluripotency. Mechanistically, as a transcriptional target of Klf4, March5 catalyses K63-linked polyubiquitination of Prkar1a, a negative regulatory subunit of PKA, to activate PKA, thereby inhibiting the Raf/MEK/ERK pathway. Moreover, March5 is able to replace a MEK/ERK inhibitor to maintain mESC pluripotency under serum-free culture conditions. In addition, March5 can partially replace the use of Klf4 for somatic cell reprogramming. Collectively, our study uncovers a role for the Klf4–March5–PKA–ERK pathway in maintaining the stemness properties of mESCs. PMID:26033541

  9. Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1.

    PubMed

    Karakasili, Eleni; Burkert-Kautzsch, Cornelia; Kieser, Anja; Sträßer, Katja

    2014-01-01

    Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that-consistent with previous data-DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII.

  10. Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1

    PubMed Central

    Zhang, Quan; Jia, Kai-Zhi; Xia, Shi-Tao; Xu, Yang-Hua; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2016-01-01

    Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering. PMID:26860895

  11. The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

    2016-01-01

    The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710. PMID:27746629

  12. SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis

    PubMed Central

    Jia, Fengjuan; Wang, Chunyan; Huang, Jinguang; Yang, Guodong; Wu, Changai; Zheng, Chengchao

    2015-01-01

    Skp1–Cullin–F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na+ homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na+ accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis. PMID:26041321

  13. ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase.

    PubMed

    Yang, Huibin; Palmbos, Phillip L; Wang, Lidong; Kim, Evelyn H; Ney, Gina M; Liu, Chao; Prasad, Jayendra; Misek, David E; Yu, Xiaochun; Ljungman, Mats; Simeone, Diane M

    2015-11-01

    Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348-588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.

  14. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system. PMID:27570077

  15. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1

    PubMed Central

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-01-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV RESISTANCE LOCUS 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546

  16. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1.

    PubMed

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-08-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV Resistance Locus 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is Constitutively Photomorphogenic 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B.

  17. DD-ligases as a potential target for antibiotics: past, present and future.

    PubMed

    Tytgat, I; Colacino, E; Tulkens, P M; Poupaert, J H; Prévost, M; Van Bambeke, F

    2009-01-01

    DD-ligases catalyze the synthesis of the D-Ala-D-Ala and D-Ala-D-Ser dipeptides or the D Ala-D-Lac depsipeptide in an early step of peptidoglycan synthesis. Their function is essential for bacterial growth and specific to bacteria, making them attractive targets for the development of novel antibiotics. This review examines the biochemical and structural features of these enzymes and presents the main families of inhibitors described so far. Over the last 20 years, 7 structures of DD-ligases have been solved by X-ray crystallography, giving a detailed view of the general topology of the active site and of the residues in the catalytic pocket that play a central role in substrate recognition. This has paved the way to the rational design of inhibitors, which can be classified as (i) analogues of substrates, (ii) analogues of the product of the reaction, (iii) analogues of the transition state, and (iv) original scaffolds discovered by screening or by rational computer-aided design. The three first strategies have led to molecules that are polar by nature and have therefore poor access to their cytosolic target. The fourth one is potentially most promising as it yields more diverse structures. The most active molecules show affinity constants in the microM range, but microbiological evaluation remains scarce (typical MIC 1-8 mg/L for the tested compounds). These data strongly suggest targeting DD-ligases is a promising approach for discovery of new antibiotics. Future research should, however, aim at finding more potent inhibitors endowed with the appropriate pharmacokinetic properties that ensure access to their intracellular target.

  18. Targeting Neddylation Pathways to Inactivate Cullin-RING Ligases for Anticancer Therapy

    PubMed Central

    Zhao, Yongchao; Morgan, Meredith A.

    2014-01-01

    Abstract Significance: Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. Recent Advances: MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. Critical Issues: In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. Future Directions: In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s. Antioxid. Redox Signal. 21, 2383–2400. PMID:24410571

  19. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

    PubMed

    Schumacher, Dominik; Helma, Jonas; Mann, Florian A; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M Cristina; Hackenberger, Christian P R; Leonhardt, Heinrich

    2015-11-01

    A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. PMID:26404067

  20. The ubiquitin ligase APC/CCdh1 puts the brakes on DNA-end resection

    PubMed Central

    Lafranchi, Lorenzo; Sartori, Alessandro A

    2015-01-01

    DNA double-strand breaks (DSBs) are highly deleterious lesions and their misrepair can promote genomic instability, a hallmark of cancer. DNA-end resection is a cell cycle-regulated mechanism that is required for the faithful repair of DSBs. We recently discovered that the anaphase-promoting complex/cyclosome-Cdh1 (APC/CCdh1) ubiquitin ligase is responsible for the timely degradation of CtBP-interacting protein (CtIP), a key DNA-end resection factor, providing a new layer of regulation of DSB repair in human cells. PMID:27308488

  1. DATEL: A Scarless and Sequence-Independent DNA Assembly Method Using Thermostable Exonucleases and Ligase.

    PubMed

    Jin, Peng; Ding, Wenwen; Du, Guocheng; Chen, Jian; Kang, Zhen

    2016-09-16

    DNA assembly is a pivotal technique in synthetic biology. Here, we report a scarless and sequence-independent DNA assembly method using thermal exonucleases (Taq and Pfu DNA polymerases) and Taq DNA ligase (DATEL). Under the optimized conditions, DATEL allows rapid assembly of 2-10 DNA fragments (1-2 h) with high accuracy (between 74 and 100%). Owing to the simple operation system with denaturation-annealing-cleavage-ligation temperature cycles in one tube, DATEL is expected to be a desirable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering. PMID:27230689

  2. RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

    PubMed

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  3. RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    PubMed Central

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K.; Menssen, Ruth; Wolf, Dieter H.; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  4. Ubiquitin ligase ATL31 functions in leaf senescence in response to the balance between atmospheric CO2 and nitrogen availability in Arabidopsis.

    PubMed

    Aoyama, Shoki; Huarancca Reyes, Thais; Guglielminetti, Lorenzo; Lu, Yu; Morita, Yoshie; Sato, Takeo; Yamaguchi, Junji

    2014-02-01

    Carbon (C) and nitrogen (N) are essential elements for metabolism, and their availability, called the C/N balance, must be tightly coordinated for optimal growth in plants. Previously, we have identified the ubiquitin ligase CNI1/ATL31 as a novel C/N regulator by screening plants grown on C/N stress medium containing excess sugar and limited N. To elucidate further the effect of C/N balance on plant growth and to determine the physiological function of ATL31, we performed C/N response analysis using an atmospheric CO2 manipulation system. Under conditions of elevated CO2 and sufficient N, plant biomass and total sugar and starch dramatically increased. In contrast, elevated CO2 with limited N did not increase plant biomass but promoted leaf chlorosis, with anthocyanin accumulation and increased senescence-associated gene expression. Similar results were obtained with plants grown in medium containing excess sugar and limited N, suggesting that disruption of the C/N balance affects senescence progression. In ATL31-overexpressing plants, promotion of senescence under disrupted CO2/N conditions was repressed, whereas in the loss-of-function mutant it was enhanced. The ATL31 gene was transcriptionally up-regulated under N deficiency and in senescent leaves, and ATL31 expression was highly correlated with WRKY53 expression, a key regulator of senescence. Furthermore, transient protoplast analysis implicated the direct activation of ATL31 expression by WRKY53, which was in accordance with the results of WRKY53 overexpression experiments. Together, these results demonstrate the importance of C/N balance in leaf senescence and the involvement of ubiquitin ligase ATL31 in the process of senescence in Arabidopsis.

  5. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. PMID:26567315

  6. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

    PubMed Central

    Koliopoulos, Marios G.; Morris-Davies, Aylin C.; Schaeffer, Veronique; Christodoulou, Evangelos; Howell, Steven; Brown, Nicholas R.; Dikic, Ivan; Rittinger, Katrin

    2013-01-01

    Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis1-5. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multi-subunit E3 ligase6. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin7. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC. PMID:24141947

  7. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

    PubMed

    Cavadini, Simone; Fischer, Eric S; Bunker, Richard D; Potenza, Alessandro; Lingaraju, Gondichatnahalli M; Goldie, Kenneth N; Mohamed, Weaam I; Faty, Mahamadou; Petzold, Georg; Beckwith, Rohan E J; Tichkule, Ritesh B; Hassiepen, Ulrich; Abdulrahman, Wassim; Pantelic, Radosav S; Matsumoto, Syota; Sugasawa, Kaoru; Stahlberg, Henning; Thomä, Nicolas H

    2016-03-31

    The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

  8. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

    NASA Technical Reports Server (NTRS)

    Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

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

    PubMed Central

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

    1995-01-01

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

  10. E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

    DOE PAGES

    Pan, Ronghui; Satkovich, John; Hu, Jianping

    2016-10-31

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

  11. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression.

    PubMed

    David, Diana; Nair, S Asha; Pillai, M Radhakrishna

    2013-01-01

    Smad ubiquitin regulatory factors (Smurfs) belong to the HECT- family of E3 ubiquitin ligases and comprise mainly of two members, Smurf1 and Smurf2. Initially, Smurfs have been implicated in determining the competence of cells to respond to TGF-β/BMP signaling pathway. Nevertheless, the intrinsic catalytic activity has extended the repertoire of Smurf substrates beyond the TGF-β/BMP super family expanding its realm further to epigenetic modifications of histones governing the chromatin landscape. Through regulation of a large number of proteins in multiple cellular compartments, Smurfs regulate diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and metastasis. As the genomic ablation of Smurfs leads to global changes in histone modifications and predisposition to a wide spectrum of tumors, Smurfs are also considered to have a novel tumor suppressor function. This review focuses on regulation network and biological functions of Smurfs in connection with its role in cancer progression. By providing a portrait of their protein targets, we intend to link the substrate specificity of Smurfs with their contribution to tumorigenesis. Since the regulation and biological functions of Smurfs are quite complex, understanding the oncogenic potential of these E3 ubiquitin ligases may facilitate the development of mechanism-based drugs in cancer treatment.

  12. Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

    PubMed Central

    Keuss, Matthew J.; Thomas, Yann; Mcarthur, Robin; Wood, Nicola T.; Knebel, Axel; Kurz, Thimo

    2016-01-01

    ABSTRACT Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression. PMID:26906416

  13. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

    PubMed

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

  14. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

    PubMed Central

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID—a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)—is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation. DOI: http://dx.doi.org/10.7554/eLife.08536.001 PMID:26393420

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

    PubMed

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

    2016-09-12

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

  16. KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase.

    PubMed

    Sambuughin, Nyamkhishig; Swietnicki, Wieslaw; Techtmann, Stephen; Matrosova, Vera; Wallace, Tarina; Goldfarb, Lev; Maynard, Ernest

    2012-05-18

    Autosomal dominant mutations in BTB and Kelch domain containing 13 protein (KBTBD13) are associated with a new type of Nemaline Myopathy (NEM). NEM is a genetically heterogeneous group of muscle disorders. Mutations causing phenotypically distinct NEM variants have previously been identified in components of muscle thin filament. KBTBD13 is a muscle specific protein composed of an N terminal BTB domain and a C terminal Kelch-repeat domain. The function of this newly identified protein in muscle remained unknown. In this study, we show that KBTBD13 interacts with Cullin 3 (Cul3) and the BTB domain mediates this interaction. Using ubiquitination assays, we determined that KBTBD13 participates in the formation of a Cul3 based RING ubiquitin ligase (Cul3-RL) capable of ubiquitin conjugation. Confocal microscopy of transiently expressed KBTBD13 revealed its co-localization with ubiquitin. Taken together, our results demonstrate that KBTBD13 is a putative substrate adaptor for Cul3-RL that functions as a muscle specific ubiquitin ligase, and thereby implicate the ubiquitin proteasome pathway in the pathogenesis of KBTBD13-associated NEM. PMID:22542517

  17. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency.

  18. RNF185, a Novel Mitochondrial Ubiquitin E3 Ligase, Regulates Autophagy through Interaction with BNIP1

    PubMed Central

    Tang, Fei; Wang, Bin; Li, Na; Wu, Yanfang; Jia, Junying; Suo, Talin; Chen, Quan; Liu, Yong-Jun; Tang, Jie

    2011-01-01

    Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy. PMID:21931693

  19. SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

    PubMed Central

    Fierer, Jacob O.; Veggiani, Gianluca; Howarth, Mark

    2014-01-01

    Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein–peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide–peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide–peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture. PMID:24639550

  20. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

    PubMed

    Cavadini, Simone; Fischer, Eric S; Bunker, Richard D; Potenza, Alessandro; Lingaraju, Gondichatnahalli M; Goldie, Kenneth N; Mohamed, Weaam I; Faty, Mahamadou; Petzold, Georg; Beckwith, Rohan E J; Tichkule, Ritesh B; Hassiepen, Ulrich; Abdulrahman, Wassim; Pantelic, Radosav S; Matsumoto, Syota; Sugasawa, Kaoru; Stahlberg, Henning; Thomä, Nicolas H

    2016-03-31

    The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN. PMID:27029275

  1. The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation.

    PubMed

    Wu, Ching-Shyi; Zou, Lee

    2016-01-01

    The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. PMID:26565033

  2. Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na(+) channel.

    PubMed

    McDonald, Fiona J; Western, Andrea H; McNeil, John D; Thomas, Brittany C; Olson, Diane R; Snyder, Peter M

    2002-09-01

    The epithelial Na(+) channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4-2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na(+) transport in epithelia. PMID:12167593

  3. The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis.

    PubMed

    Čajánek, Lukas; Glatter, Timo; Nigg, Erich A

    2015-05-01

    Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here, we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a new interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitylation of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a new and important element in the control of centriole homeostasis.

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

    PubMed Central

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

    2015-01-01

    In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins. PMID:25043012

  5. A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA.

    PubMed

    Wang, Yonghong; He, Xiaoxiao; Wang, Kemin; Ni, Xiaoqi

    2010-05-15

    A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5'-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 10(3) and 10(7) cfu/mL.

  6. Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase.

    PubMed Central

    Higgins, N P; Geballe, A P; Cozzarelli, N R

    1979-01-01

    Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA. Images PMID:375192

  7. CUL4A ubiquitin ligase: a promising drug target for cancer and other human diseases

    PubMed Central

    Sharma, Puneet; Nag, Alo

    2014-01-01

    The ability of cullin 4A (CUL4A), a scaffold protein, to recruit a repertoire of substrate adaptors allows it to assemble into distinct E3 ligase complexes to mediate turnover of key regulatory proteins. In the past decade, a considerable wealth of information has been generated regarding its biology, regulation, assembly, molecular architecture and novel functions. Importantly, unravelling of its association with multiple tumours and modulation by viral proteins establishes it as one of the key proteins that may play an important role in cellular transformation. Considering the role of its substrate in regulating the cell cycle and maintenance of genomic stability, understanding the detailed aspects of these processes will have significant consequences for the treatment of cancer and related diseases. This review is an effort to provide a broad overview of this multifaceted ubiquitin ligase and addresses its critical role in regulation of important biological processes. More importantly, its tremendous potential to be exploited for therapeutic purposes has been discussed. PMID:24522884

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

    PubMed Central

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

    2015-01-01

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882. PMID:26217795

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

    PubMed

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

    2015-12-01

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

  10. A conserved role for the ARC1 E3 ligase in Brassicaceae self-incompatibility.

    PubMed

    Indriolo, Emily; Goring, Daphne R

    2014-01-01

    Ubiquitination plays essential roles in the regulation of many processes in plants including pollen rejection in self-incompatible species. In the Brassicaceae (mustard family), self-incompatibility drives the rejection of self-pollen by preventing pollen hydration following pollen contact with the stigmatic surface. Self-pollen is recognized by a ligand-receptor pair: the pollen S-locus cysteine rich/S-locus protein 11 (SCR/SP11) ligand and the pistil S receptor kinase (SRK). Following self-pollen contact, the SCR/SP11 ligand on the pollen surface binds to SRK on the pistil surface, and the SRK-activated signaling pathway is initiated. This pathway includes the armadillo repeat containing 1 (ARC1) protein, a member of the plant U-box (PUB) family of E3 ubiquitin ligases. ARC1 is a functional E3 ligase and is required downstream of SRK for the self-incompatibility response. This mini review highlights our recent progress in establishing ARC1's conserved role in self-pollen rejection in Brassica and Arabidopsis species and discusses future research directions in this field. PMID:24847339

  11. Genomic and functional analysis of the E3 ligase PARK2 in glioma

    PubMed Central

    Lin, De-Chen; Xu, Liang; Chen, Ye; Yan, Haiyan; Hazawa, Masaharu; Doan, Ngan; Said, Jonathan W.; Ding, Ling-Wen; Liu, Li-Zhen; Yang, Henry; Yu, Shizhu; Kahn, Michael; Yin, Dong; Koeffler, H. Phillip

    2015-01-01

    PARK2 (PARKIN) is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of Parkinsonism and human malignancies, and its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, while depletion of PARK2 promoted cell proliferation. PARK2 attenuated both Wnt- and EGF-stimulated pathways through down-regulating the intracellular level of β-catenin and EGFR. Notably, PARK2 physically interacted with both β-catenin and EGFR. We further found that PARK2 promoted the ubiquitination of these two proteins in an E3 ligase activity dependent manner. Finally, inspired by these newly identified tumor suppressive functions of PARK2, we tested and proved that combination of small-molecule inhibitors targeting both Wnt-β-catenin and EGFR-AKT pathways synergistically impaired glioma cell viability. Together, our findings uncover novel cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. PMID:25877876

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

    PubMed Central

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

    2016-01-01

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

  13. Upregulation of the E3 ligase NEDD4-1 by Oxidative Stress Degrades IGF-1 Receptor Protein in Neurodegeneration

    PubMed Central

    Kwak, Young-Don; Wang, Bin; Li, Jing Jing; Wang, Ruishan; Deng, Qiyue; Diao, Shiyong; Chen, Yaomin; Xu, Raymond; Masliah, Eliezer; Xu, Huaxi; Sung, Jung-Joon

    2012-01-01

    The importance of ubiquitin E3 ligases in neurodegeneration is being increasingly recognized. The crucial role of NEDD4-1 in neural development is well appreciated; however, its role in neurodegeneration remains unexplored. Herein, we report increased NEDD4-1 expression in the degenerated tissues of several major neurodegenerative diseases. Moreover, its expression is upregulated in cultured neurons in response to various neurotoxins, including zinc and hydrogen superoxide, via transcriptional activation likely mediated by the reactive oxygen species (ROS)-responsive FOXM1B. Reduced protein levels of the insulin-like growth factor receptor (IGF-1Rβ) were observed as a consequence of upregulated NEDD4-1 via the ubiquitin-proteasome system. Overexpression of a familial mutant form of superoxide dismutase 1 (SOD1) (G93A) in neuroblastoma cells resulted in a similar reduction of IGF-1Rβ protein. This inverse correlation between NEDD4-1 and IGF-1Rβ was also observed in the cortex and spinal cords of mutant (G93A) SOD1 transgenic mice at a presymptomatic age, which was similarly induced by in vivo-administered zinc in wild-type C57BL/6 mice. Furthermore, histochemistry reveals markedly increased NEDD4-1 immunoreactivity in the degenerating/degenerated motor neurons in the lumbar anterior horn of the spinal cord, suggesting a direct causative role for NEDD4-1 in neurodegeneration. Indeed, downregulation of NEDD4-1 by shRNA or overexpression of a catalytically inactive form rescued neurons from zinc-induced cell death. Similarly, neurons with a NEDD4-1 haplotype are more resistant to apoptosis, largely due to expression of higher levels of IGF-1Rβ.Together, our work identifies a novel molecular mechanism for ROS-upregulated NEDD4-1 and the subsequently reduced IGF-1Rβ signaling in neurodegeneration. PMID:22875931

  14. The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

    PubMed

    Cao, Juxiang; Dai, Xiangpeng; Wan, Lixin; Wang, Hongshen; Zhang, Jinfang; Goff, Philip S; Sviderskaya, Elena V; Xuan, Zhenyu; Xu, Zhixiang; Xu, Xiaowei; Hinds, Philip; Flaherty, Keith T; Faller, Douglas V; Goding, Colin R; Wang, Yongjun; Wei, Wenyi; Cui, Rutao

    2015-09-01

    The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma. PMID:26329581

  15. Comparison of a Ligase Chain Reaction-Based Assay and Cell Culture for Detection of Pharyngeal Carriage of Chlamydia trachomatis

    PubMed Central

    Winter, Andrew J.; Gilleran, Gerry; Eastick, Kirstine; Ross, Jonathan D. C.

    2000-01-01

    In 264 genitourinary medicine clinic attenders reporting recent fellatio, the prevalence of pharyngeal Chlamydia trachomatis determined by an expanded standard including cell culture and two in-house PCR tests was 1.5% in 194 women and zero in 70 men. The ligase chain reaction (Abbott LCx) had a specificity of 99.2% and a positive predictive value of 60%. PMID:10970416

  16. Universal Labeling of 5′-Triphosphate RNAs by Artificial RNA Ligase Enzyme with Broad Substrate Specificity

    PubMed Central

    Haugner, John C.; Seelig, Burckhard

    2013-01-01

    An artificial RNA ligase specific to RNA with a 5′-triphosphate (PPP-RNA) exhibits broad sequence specificity on model substrates and secondary siRNAs with direct applications in the identification of PPP-RNAs through sequencing. PMID:23851643

  17. A simple template-dependent ligase ribozyme as the RNA replicase emerging first in the RNA world.

    PubMed

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  18. Regulation of 4CL, encoding 4-coumarate: coenzyme A ligase, expression in kenaf under diverse stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned the full length 4CL ortholog encoding 4-coumarate: coenzymeA ligase from kenaf (Hibiscus cannabiuns) using degenerate primers and RACE (rapid amplification of cDNA ends) systems. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic ac...

  19. Sorghum Brown midrib 2 (Bmr2) gene encodes the major 4-coumarate Coenzyme A ligase involved in lignin synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Successful modification of plant cell wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but can be very challenging when the target genes are members of multigene families. 4-Coumarate:CoA ligase (4CL) catalyzes the formatio...

  20. Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

    PubMed Central

    Gu, Huiqiong; Yoshinari, Shigeo; Ghosh, Raka; Ignatochkina, Anna V.; Gollnick, Paul D.; Murakami, Katsuhiko S.; Ho, C. Kiong

    2016-01-01

    An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl–AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5′-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl–AMP complex. PMID:26896806

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Identifying the trigger of c-IAPs: structural and functional characterization of CARD-mediated modulation of ubiquitin ligase activity.

    PubMed

    Oetjen, Karolyn A; Duckett, Colin S

    2011-06-10

    In this issue of Molecular Cell, Lopez et al. (2011) examine the caspase-recruitment domain (CARD) of c-IAP1 to reveal an intriguing mechanism in which conformational changes of the CARD determine c-IAP1's ubiquitin ligase activity, with implications for regulation of cell proliferation and survival by the IAPs.

  3. A Simple Template-Dependent Ligase Ribozyme as the RNA Replicase Emerging First in the RNA World

    NASA Astrophysics Data System (ADS)

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  4. The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding*

    PubMed Central

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

    2015-01-01

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity. PMID:26224628

  5. Competing E3 Ubiquitin Ligases Determine Circadian Period by Regulated Degradation of CRY in Nucleus and Cytoplasm

    PubMed Central

    Yoo, Seung-Hee; Mohawk, Jennifer A.; Siepka, Sandra M.; Shan, Yongli; Huh, Seong Kwon; Hong, Hee-Kyung; Kornblum, Izabela; Kumar, Vivek; Koike, Nobuya; Xu, Ming; Nussbaum, Justin; Liu, Xinran; Chen, Zheng; Chen, Zhijian J.; Green, Carla B.; Takahashi, Joseph S.

    2013-01-01

    SUMMARY Period determination in the mammalian circadian clock involves the turnover rate of the repressors, CRY and PER. Here we show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate (G149E) missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss-of-function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm, but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals. PMID:23452855

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

    PubMed

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

    2015-09-11

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

  7. Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

    PubMed

    Wiśniewski, Marcin; Lapiński, Maciej; Zdziarska, Anna; Długosz, Ewa; Bąska, Piotr

    2014-08-01

    Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor. PMID

  8. CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase.

    PubMed

    Shibata, Etsuko; Dar, Ashraf; Dutta, Anindya

    2014-08-15

    Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4(Cdt2), specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4(Cdt2)-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.

  9. Downregulation of E3 Ubiquitin Ligases and Mitophagy-Related Genes in Skeletal Muscle of Physically Inactive, Frail Older Women: A Cross-Sectional Comparison

    PubMed Central

    Addison, Odessa; Brunker, Lucille; Hopkins, Paul N.; McClain, Donald A.; LaStayo, Paul C.; Marcus, Robin L.

    2014-01-01

    Background and Objectives. Reduced lean mass and physical function is a characteristic of frailty. However, it is currently unknown if proteolysis through the E3 ubiquitin ligases and the autophagic lysosomal pathway is dysregulated in inactive frail older women. The purpose of this study was to determine the expression of key markers of ubiquitin-mediated and autophagic lysosomal proteolysis in inactive (N = 7) compared with active (N = 7) older women. Methods. Strength, mobility, leg lean mass, and physical activity assessment were used to characterize activity levels and frailty in older women. Vastus lateralis biopsies were collected after an overnight fast and were assessed for gene and protein targets related to E3 ubiquitin ligases and the autophagic lysosomal system. Results. We found that AMP-activated protein kinase alpha (Thr172) was increased (p = .045), and forkhead box O3A (FOXO3A) gene expression (p = .047) was lower in inactive frail older women. Foxo3a (Ser253), Beclin1 (Ser93/96), and class III phosphatidylinositol-3-kinase (VPS34) protein expression were not different between the groups (p > .05). Neural precursor cell-expressed developmentally downregulated protein 4, muscle ring finger 1, muscle atrophy F-box, and the autophagy/mitophagy gene expression markers, Beclin1, autophagy-related-7, BCL2/adenovirus E1B 19kDa interacting protein 3 (BNIP3), dynamin-related protein 1, and Parkinson protein 2 (PARKIN) were lower in inactive frail older women (p < .05). Autophagy/mitophagy markers were positively correlated with the 6-minute walk and leg lean mass (p < .05). Conclusions. We conclude that physical inactivity in frail older women is associated with a downregulation of ubiquitin-mediated and autophagic lysosomal skeletal muscle gene expression, perhaps related to low muscle mass and poor physical function. PMID:24526667

  10. Targeting the Oncogenic E3 Ligase Skp2 in Prostate and Breast Cancer Cells with a Novel Energy Restriction-Mimetic Agent

    PubMed Central

    Wei, Shuo; Chu, Po-Chen; Chuang, Hsiao-Ching; Hung, Wen-Chun; Kulp, Samuel K.; Chen, Ching-Shih

    2012-01-01

    Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of β-transducin repeat-containing protein (β-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this β-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets β-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif 412SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and β-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of β-TrCP reduced the expression of Sp1, a β-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-β-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a “druggable” target for this novel ERMA. PMID:23071779

  11. The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for GLUTAMINE DUMPER1-Induced Amino Acid Secretion in Arabidopsis1[C][W][OA

    PubMed Central

    Pratelli, Réjane; Guerra, Damian D.; Yu, Shi; Wogulis, Mark; Kraft, Edward; Frommer, Wolf B.; Callis, Judy; Pilot, Guillaume

    2012-01-01

    Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1’s role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells. PMID:22291198

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

    PubMed

    Cheng, Jie; Guggino, William

    2013-01-01

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

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

    PubMed Central

    ZeRuth, Gary T.; Williams, Jason G.; Cole, Yasemin C.; Jetten, Anton M.

    2015-01-01

    The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases. PMID:26147758

  14. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  15. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV.

    PubMed

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin; Ding, Nan; Qi, Yongmei; Zhang, Yingmei; Wang, Jufang; Huang, Dejun

    2015-09-01

    Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1-8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

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

    PubMed Central

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

    2015-01-01

    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

  17. Both Ubiquitin Ligases FBXW8 and PARK2 Are Sequestrated into Insolubility by ATXN2 PolyQ Expansions, but Only FBXW8 Expression Is Dysregulated

    PubMed Central

    Halbach, Melanie Vanessa; Stehning, Tanja; Damrath, Ewa; Jendrach, Marina; Şen, Nesli Ece; Başak, A. Nazlı; Auburger, Georg

    2015-01-01

    The involvement of the ubiquitin-proteasome system (UPS) in the course of various age-associated neurodegenerative diseases is well established. The single RING finger type E3 ubiquitin-protein ligase PARK2 is mutated in a Parkinson’s disease (PD) variant and was found to interact with ATXN2, a protein where polyglutamine expansions cause Spinocerebellar ataxia type 2 (SCA2) or increase the risk for Levodopa-responsive PD and for the motor neuron disease Amyotrophic lateral sclerosis (ALS). We previously reported evidence for a transcriptional induction of the multi-subunit RING finger Skp1/Cul/F-box (SCF) type E3 ubiquitin-protein ligase complex component FBXW8 in global microarray profiling of ATXN2-expansion mouse cerebellum and demonstrated its role for ATXN2 degradation in vitro. Now, we documented co-localization in vitro and co-immunoprecipitations both in vitro and in vivo, which indicate associations of FBXW8 with ATXN2 and PARK2. Both FBXW8 and PARK2 proteins are driven into insolubility by expanded ATXN2. Whereas the FBXW8 transcript upregulation by ATXN2- expansion was confirmed also in qPCR of skin fibroblasts and blood samples of SCA2 patients, a FBXW8 expression dysregulation was not observed in ATXN2-deficient mice, nor was a PARK2 transcript dysregulation observed in any samples. Jointly, all available data suggest that the degradation of wildtype and mutant ATXN2 is dependent on FBXW8, and that ATXN2 accumulation selectively modulates FBXW8 levels, while PARK2 might act indirectly through FBXW8. The effects of ATXN2-expansions on FBXW8 expression in peripheral tissues like blood may become useful for clinical diagnostics. PMID:25790475

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

    PubMed

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

    2015-11-16

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

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

    PubMed

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

    2015-11-16

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

  20. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

    PubMed

    Laedermann, Cédric J; Cachemaille, Matthieu; Kirschmann, Guylène; Pertin, Marie; Gosselin, Romain-Daniel; Chang, Isabelle; Albesa, Maxime; Towne, Chris; Schneider, Bernard L; Kellenberger, Stephan; Abriel, Hugues; Decosterd, Isabelle

    2013-07-01

    Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain. PMID:23778145

  1. The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization*

    PubMed Central

    Cubillos-Rojas, Monica; Amair-Pinedo, Fabiola; Peiró-Jordán, Roser; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2014-01-01

    The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292–2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling. PMID:24722987

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

    PubMed

    Ling, Qihua; Jarvis, Paul

    2015-10-01

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

  3. Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme.

    PubMed

    Tuzlakoglu Ozturk, Merve; Akbulut, Nagihan; Issever Ozturk, Saliha; Gumusel, Fusun

    2013-09-01

    Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5-7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co(+2), Cu(2+), Fe(2+), Fe(3+), Ni(+2), and Zn(+2) caused significant inhibition in enzyme activity, which was not affected by Na(+), Mg(2+), Li(+), and Ba(2+). The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca(2+) ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.

  4. Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.

    PubMed Central

    Wiedmann, M; Barany, F; Batt, C A

    1993-01-01

    A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h. Images PMID:8368859

  5. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.

    PubMed

    Lewcock, Joseph W; Genoud, Nicolas; Lettieri, Karen; Pfaff, Samuel L

    2007-11-21

    To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.

  6. Bisubstrate Inhibitors of Biotin Protein Ligase in Mycobacterium tuberculosis Resistant to Cyclonucleoside Formation

    PubMed Central

    2013-01-01

    Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is the leading cause of bacterial infectious disease mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for Mtb survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant Mtb strains. Here we present the design, synthesis, and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5′ oxygen atom with carbon as well as incorporation of a bulky group on the nucleobase to prevent the required syn-conformation necessary for proper alignment of N-3 with C-5′. PMID:24363833

  7. Cortical dynamics during cell motility are regulated by CRL3(KLHL21) E3 ubiquitin ligase.

    PubMed

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

    2016-01-01

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions. PMID:27641145

  8. Structural and functional insights into the E3 ligase, RNF126

    PubMed Central

    Krysztofinska, Ewelina M.; Martínez-Lumbreras, Santiago; Thapaliya, Arjun; Evans, Nicola J.; High, Stephen; Isaacson, Rivka L.

    2016-01-01

    RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm. PMID:27193484

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

    PubMed Central

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

    2016-01-01

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

  10. Cortical dynamics during cell motility are regulated by CRL3(KLHL21) E3 ubiquitin ligase.

    PubMed

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

    2016-09-19

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

  11. Rational optimization of the DSL ligase ribozyme with GNRA/receptor interacting modules.

    PubMed

    Ishikawa, Junya; Matsumura, Shigeyoshi; Jaeger, Luc; Inoue, Tan; Furuta, Hiroyuki; Ikawa, Yoshiya

    2009-10-15

    The DSL ribozyme is a class of artificial ligase ribozymes with a highly modular architecture, which catalyzes template-directed RNA ligation on a helical substrate module that can be either covalently connected (cis-DSL) or physically separated (trans-DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively. In this study, we have rationally designed and analyzed the catalytic and self-assembly properties of several trans-DSL ribozymes with different sets of natural and artificial GNRA-receptor clamps. Two variants newly designed in this study showed significantly enhanced catalytic properties with respect of the original trans-DSL construct. While this work allows dissection of the turnover and catalytic properties of the trans-DSL ribozyme, it also emphasizes the remarkable modularity of RNA tertiary structure for nano-construction of complex functions.

  12. Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction.

    PubMed

    Batt, C A; Wagner, P; Wiedmann, M; Luo, J; Gilbert, R

    1994-04-01

    A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3' end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos 530 and the entire assay including DNA isolation can be completed within 8 h. PMID:7912052

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

    PubMed Central

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

    2012-01-01

    S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family. It is a component of the SCF E3 ubiquitin ligase complex. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation, including cyclin-dependent kinase inhibitor p27. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers. This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence, cancer progression, and metastasis, as well as the therapeutic potential of targeting Skp2 for human cancer treatment. PMID:22200179

  14. Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization.

    PubMed

    Yau, Richard G; Peng, Yutian; Valiathan, Rajeshwari R; Birkeland, Shanda R; Wilson, Thomas E; Weisman, Lois S

    2014-03-10

    Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2, binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism that detaches myosin V from select cargoes.

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

    PubMed Central

    Li, Wei

    2010-01-01

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

  16. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    PubMed Central

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  17. The E3 ligase ube3a is required for learning in Drosophila melanogaster.

    PubMed

    Chakraborty, Moumita; Paul, Blesson K; Nayak, Tanmoyita; Das, Aniruddha; Jana, Nihar R; Bhutani, Supriya

    2015-06-19

    Angelman syndrome and autism are neurodevelopmental disorders linked to mutations and duplications of an E3 ligase called ube3a respectively. Since cognitive deficits and learning disabilities are hallmark symptoms of both these disorders, we investigated a role for dube3a in the learning ability of flies using the aversive phototaxis suppression assay. We show that down and up-regulation of dube3a are both detrimental to learning in larvae and adults. Using conditional gene expression we found that dube3a is required for normal brain development and during adulthood. Furthermore, we suggest that dube3a could be interacting with other learning and memory genes such as derailed. Along with firmly establishing dube3a as a gene that is required for learning, our work also opens avenues for further understanding the role played by this gene in brain development and behavior. PMID:25935478

  18. Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

    PubMed Central

    Song, Yunke; Zhang, Yi; Wang, Tza-Huei

    2014-01-01

    Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. PMID:23239594

  19. An immobilized biotin ligase: surface display of Escherichia coli BirA on Saccharomyces cerevisiae.

    PubMed

    Parthasarathy, Ranganath; Bajaj, Jitin; Boder, Eric T

    2005-01-01

    The Escherichia coli biotin ligase enzyme BirA has been extensively used in recent years to generate site-specifically biotinylated proteins via a biotin acceptor peptide tag. In the present study, BirA was displayed for the first time on the yeast Saccharomyces cerevisiae using the Aga1p-Aga2p platform and assayed using a peptide-tagged protein as the substrate. The enzyme is fully functional and resembles the soluble form in many of its properties, but the yeast-displayed enzyme demonstrates stability and reusability on the time scale of weeks. Thus, the yeast-displayed BirA system represents a facile and highly economical alternative for producing site-specifically biotinylated proteins.

  20. β-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1.

    PubMed

    Suriben, Rowena; Kaihara, Kelly A; Paolino, Magdalena; Reichelt, Mike; Kummerfeld, Sarah K; Modrusan, Zora; Dugger, Debra L; Newton, Kim; Sagolla, Meredith; Webster, Joshua D; Liu, Jinfeng; Hebrok, Matthias; Dixit, Vishva M

    2015-12-01

    A variety of signals finely tune insulin secretion by pancreatic β cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in β cells is critical for insulin secretion. Mice lacking COP1 in β cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human β-cell pathophysiology. In normal β cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.

  1. Development of ligase-assisted spacer addition for the measurement of microsatellites.

    PubMed

    Brockhurst, V; Barnard, R; Wolter, L; Giffard, P; Timms, P

    2001-07-01

    Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method. PMID:11464526

  2. The E3 ligase Itch and deubiquitinase Cyld co-operatively regulate Tak1 and inflammation

    PubMed Central

    Ahmed, Neesar; Zeng, Minghui; Sinha, Indrajit; Polin, Lisa; Wei, Wei-Zen; Rathinam, Chozhavendan; Flavell, Richard; Massoumi, Ramin; Venuprasad, K

    2011-01-01

    Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells. PMID:22057290

  3. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  4. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  5. An E3 ligase complex regulates SET-domain polycomb group protein activity in Arabidopsis thaliana

    PubMed Central

    Jeong, Cheol Woong; Roh, Hyungmin; Dang, Tuong Vi; Choi, Yang Do; Fischer, Robert L.; Lee, Jong Seob; Choi, Yeonhee

    2011-01-01

    Transcriptional repression via methylation of histone H3 lysine 27 (H3K27) by the polycomb repressive complex 2 (PRC2) is conserved in higher eukaryotes. The Arabidopsis PRC2 controls homeotic gene expression, flowering time, and gene imprinting. Although downstream target genes and the regulatory mechanism of PRC2 are well understood, much less is known about the significance of posttranslational regulation of PRC2 protein activity. Here, we show the posttranslational regulation of CURLY LEAF (CLF) SET-domain polycomb group (PcG) protein by the F-box protein, UPWARD CURLY LEAF1 (UCL1). Overexpression of UCL1 generates mutant phenotypes similar to those observed in plants with a loss-of-function mutation in the CLF gene. Leaf curling and early flowering phenotypes of UCL1 overexpression mutants, like clf mutants, are rescued by mutations in the AGAMOUS and FLOWERING LOCUS T genes, which is consistent with UCL1 and CLF functioning in the same genetic pathway. Overexpression of UCL1 reduces the level of CLF protein and alters expression and H3K27 methylation of CLF-target genes in transgenic plants, suggesting that UCL1 negatively regulates CLF. Interaction of UCL1 with CLF was detected in plant nuclei and in the yeast two-hybrid system. The UCL1 F-box binds in vivo to components of the E3 ligase complex, which ubiquitylate proteins that are subsequently degraded via the ubiquitin-26S proteasome pathway. Taken together, these results demonstrate the posttranslational regulation of the CLF SET-domain PcG activity by the UCL1 F-box protein in the E3 ligase complex. PMID:21518870

  6. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

    PubMed

    Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

    2011-04-01

    During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms

  7. SGR9, a RING type E3 ligase, modulates amyloplast dynamics important for gravity sensing.

    NASA Astrophysics Data System (ADS)

    Morita, Miyo T.; Nakamura, Moritaka; Tasaka, Masao

    Gravitropism is triggered when the directional change of gravity is sensed in the specific cells, called statocytes. In higher plants, statocytes contain sinking heavier amyloplasts which are particular plastids accumulating starch granules. The displacement of amyloplasts within the statocytes is thought to be the initial event of gravity perception. We have demonstrated that endodermal cells are most likely to be the statocytes in Arabidop-sis shoots. Live cell imaging of the endodermal cell of stem has shown that most amyloplasts are sediment to the direction of gravity but they are not static. Several amyloplasts move dynamically in an actin filament (F-actin) dependent manner. In the presence of actin poly-merization inhibitor, all amyloplasts become static and sediment to the direction of gravity. In addition, stems treated with the inhibitor can exhibit gravitropism. These results suggest that F-actin-dependent dynamic movement of amyloplasts is not essential for gravity sensing. sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, dynamic amyloplast movement was predominantly observed and amyloplasts did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization re-stored both gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiq-uitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylo-plasts. SGR9 positively act on amyloplasts sedimentation, probably by releasing amyloplasts from F-actin. SGR9 that is localized to amyloplast, possibly degrades unknown substrates by its E3 ligase activity, and this might promote release of amyloplasts from F-actin.

  8. The Functions of the HIV1 protein Vpr and its action through the DCAF1•DDB1•Cullin4 ubiquitin ligase

    PubMed Central

    Casey, Laurieann; Wen, Xiaoyun; de Noronha, Carlos M.C.

    2010-01-01

    Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1•Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities. PMID:20347598

  9. Rational Redesign of the 4-Chlorobenzoate Binding Site of 4-Chlorobenzoate: Coenzyme A Ligase for Expanded Substrate Range#%

    PubMed Central

    Wu, Rui; Reger, Albert S.; Cao, Jian; Gulick, Andrew M.; Dunaway-Mariano, Debra

    2014-01-01

    Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid by-product formed can be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid: CoA ligase must be an effective catalyst with the chlorinated benzoic acid. The present study, which focuses on the 4-chlorobenzoate: CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp strain ALP83, was carried-out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (so to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against ring meta-C(H). The structural basis for the ~100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G

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

    PubMed

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

    2015-09-01

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

  11. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-09-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.

  12. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

  13. Negishi cross-coupling enabled synthesis of novel NAD(+)-dependent DNA ligase inhibitors and SAR development.

    PubMed

    Murphy-Benenato, Kerry E; Gingipalli, Lakshmaiah; Boriack-Sjodin, P Ann; Martinez-Botella, Gabriel; Carcanague, Dan; Eyermann, Charles J; Gowravaram, Madhu; Harang, Jenna; Hale, Michael R; Ioannidis, Georgine; Jahic, Harris; Johnstone, Michele; Kutschke, Amy; Laganas, Valerie A; Loch, James T; Miller, Matthew D; Oguto, Herbert; Patel, Sahil Joe

    2015-11-15

    Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.

  14. A tail of two sites: a bipartite mechanism for recognition of notch ligands by mind bomb E3 ligases.

    PubMed

    McMillan, Brian J; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C

    2015-03-01

    Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

  15. The highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular SCF ubiquitin ligase complex.

    PubMed

    Sperling, Karin M; Schwantes, Astrid; Schnierle, Barbara S; Sutter, Gerd

    2008-05-10

    The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF complex formation might be important for the viral life cycle.

  16. The tumour antigen PRAME is a subunit of a Cul2 ubiquitin ligase and associates with active NFY promoters

    PubMed Central

    Costessi, Adalberto; Mahrour, Nawel; Tijchon, Esther; Stunnenberg, Rieka; Stoel, Marieke A; Jansen, Pascal W; Sela, Dotan; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan W; Conaway, Ronald C; Stunnenberg, Hendrik G

    2011-01-01

    The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation. PMID:21822215

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

    SciTech Connect

    Yunus, Ali A.; Lima, Christopher D.

    2010-01-12

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Unusually divergent 4-coumarate:CoA-ligases from Ruta graveolens L.

    PubMed

    Endler, Alexander; Martens, Stefan; Wellmann, Frank; Matern, Ulrich

    2008-07-01

    Most angiosperms encode a small family of 4-coumarate:CoA-ligases (4CLs) activating hydroxycinnamic acids for lignin and flavonoid pathways. The common rue, Ruta graveolens L., additionally produces coumarins by cyclization of the 4-coumaroyl moiety, possibly involving the CoA-ester, as well as acridone and furoquinoline alkaloids relying on (N-methyl)anthraniloyl-CoA as the starter substrate for polyketide synthase condensation. The accumulation of alkaloids and coumarins, but not flavonoids, was enhanced in Ruta graveolens suspension cultures upon the addition of fungal elicitor. Total RNA of elicitor-treated Ruta cells was used as template for RT-PCR amplification with degenerate oligonucleotide primers inferred from conserved motifs in AMP-binding proteins, and two full-size cDNAs were generated through RACE and identified as 4-coumarate:CoA-ligases, Rg4CL1 and Rg4CL2, by functional expression in yeast cells. The recombinant enzymes differed considerably in their preferential affinities to cinnamate (Rg4CL1) or ferulate (RgCL2) besides 4-coumarate, but did not activate hydroxybenzoic or (N-methyl)anthranilic acid. Most notably, the Rg4CL1 polypeptide included an N-terminal extension suggesting a chloroplast transit peptide. The genes were cloned and revealed four exons, separated by 1056, 94 and 54 bp introns for RgCL1, while Rg4CL2 was composed of five exons interupted by four introns from 113 to 350 bp, and the divergent heritage of these genes was substantiated by phylogenetic analysis. Both genes were expressed in shoot, leaf and flower tissues of adult Ruta plants with preference in shoot and flower, whereas negligible expression occurred in the root. However, Rg4CL1 was expressed much stronger in the flower, while Rg4CL2 was expressed mostly in the shoot. Furthermore, considerable transient induction of only Rg4CL1 was observed upon elicitation of Ruta cells, which seems to support a role of Rg4CL1 in coumarin biosynthesis.

  1. Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

    PubMed Central

    Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D.; Malinsky, Sophie; Bétermier, Mireille

    2011-01-01

    During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new

  2. Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities

    PubMed Central

    2015-01-01

    Background In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. Results In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. Conclusion A case study demonstrated the effectiveness of the

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

    PubMed

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

    2013-01-01

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

  4. Fbxw5 suppresses nuclear c-Myb activity via DDB1-Cul4-Rbx1 ligase-mediated sumoylation

    SciTech Connect

    Kanei-Ishii, Chie; Nomura, Teruaki; Egoh, Ayako; Ishii, Shunsuke

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Fbxw5 enhances sumoylation of c-Myb. Black-Right-Pointing-Pointer The DDB1-Cul4A-Rbx1 complex mediates c-Myb sumoylation. Black-Right-Pointing-Pointer The Fbxw5-DDB1-Cul4A-Rdx1 complex is a dual SUMO/ubiquitin ligase. Black-Right-Pointing-Pointer Fbxw5 suppresses the c-Myb trans-activating capacity. -- Abstract: The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling. In this process, Fbxw7{alpha}, the F-box protein of the SCF complex, binds to c-Myb via its C-terminal WD40 domain, and induces the ubiquitination of c-Myb. Here, we report that Fbxw5, another F-box protein, enhances sumoylation of nuclear c-Myb. Fbxw5 enhanced c-Myb sumoylation via the DDB1-Cul4A-Rbx1 complex. Since the Fbxw5-DDB1-Cul4A-Rbx1 complex was shown to act as a ubiquitin ligase for tumor suppressor TSC2, our results suggest that this complex can function as a dual SUMO/ubiquitin ligase. Fbxw5, which is localized to both nucleus and cytosol, enhanced sumoylation of nuclear c-Myb and induced the localization of c-Myb to nuclear dot-like domains. Co-expression of Fbxw5 suppressed the trans-activation of c-myc promoter by wild-type c-Myb, but not by v-Myb, which lacks the sumoylation sites. These results suggest that multiple E3 ligases suppress c-Myb activity through sumoylation or ubiquitination, and that v-Myb is no longer subject to these negative regulations.

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

    PubMed Central

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

    2014-01-01

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

  6. The structural basis for partitioning of the XRCC1/DNA ligase III-[alpha] BRCT-mediated dimer complexes

    SciTech Connect

    Cuneo, Matthew J.; Gabel, Scott A.; Krahn, Joseph M.; Ricker, Melissa A.; London, Robert E.

    2011-11-17

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

  7. The Structural Basis for Partitioning of the XRCC1/DNA Ligase III-alpha BRCT-mediated Dimer Complexes

    SciTech Connect

    M Cuneo; S Gabel; J Krahn; M Ricker; R London

    2011-12-31

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

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

    PubMed Central

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

    2016-01-01

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

  9. Positive regulation of rice RING E3 ligase OsHIR1 in arsenic and cadmium uptakes.

    PubMed

    Lim, Sung Don; Hwang, Jin Gyu; Han, A Reum; Park, Yong Chan; Lee, Chanhui; Ok, Yong Sik; Jang, Cheol Seong

    2014-07-01

    The metalloid arsenic (As) and the heavy metal cadmium (Cd) are ubiquitously found at low concentrations in the earth. High concentrations of these elements in the soil and crops are severely dangerous to human health. We attempted to retrieve the RING E3 ubiquitin ligase gene for regulating As and Cd uptakes via the ubiquitin 26S proteasome system. Semi-quantitative reverse transcription polymerase chain reaction was conducted for a total of 47 Oryza sativa RING finger protein (OsRFP) genes to assess their expression patterns when exposed to As and Cd treatments. We identified one gene Oryza sativa heavy metal induced RING E3 ligase 1 (OsHIR1), which was significantly upregulated with both treatments. A yeast hybrid screen and a bimolecular fluorescence complementation assay showed that OsHIR1 clearly interacts with 5 substrate proteins, including tonoplast intrinsic protein 4;1 (OsTIP4;1) in the plasma membrane. In addition, OsHIR1 strongly degraded the protein level of OsTIP4;1 via the ubiquitin 26S proteasome system. Heterogeneous overexpression of OsHIR1 in Arabidopsis exhibited As- and Cd-insensitive phenotypes and resulted in decreased As and Cd accumulation in the shoots and roots, relative to the control. Herein, we report the novel finding that the OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptakes. PMID:24664473

  10. Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases*

    PubMed Central

    Canning, Peter; Cooper, Christopher D. O.; Krojer, Tobias; Murray, James W.; Pike, Ashley C. W.; Chaikuad, Apirat; Keates, Tracy; Thangaratnarajah, Chancievan; Hojzan, Viktorija; Marsden, Brian D.; Gileadi, Opher; Knapp, Stefan; von Delft, Frank; Bullock, Alex N.

    2013-01-01

    Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design. PMID:23349464

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

    PubMed

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

    2011-12-13

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

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

    PubMed Central

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

    2011-01-01

    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL–LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL–LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism. PMID:22109552

  13. Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

    PubMed Central

    Manjarrez, Jacob R.; Sun, Liang; Prince, Thomas; Matts, Robert L.

    2014-01-01

    The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes. PMID:24608665

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

    PubMed Central

    2011-01-01

    Background Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin. Results After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex. Conclusions We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins. PMID:21798009

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

    PubMed

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

    2016-08-15

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

  16. Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase.

    PubMed

    He, Xiaoxiao; Ni, Xiaoqi; Wang, Yonghong; Wang, Kemin; Jian, Lixin

    2011-01-15

    An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.

  17. Binding and Recognition in the Assembly of an Active BRCA1/BARD1 Ubiquitin-Ligase Complex

    SciTech Connect

    Brzovic, Peter S.; Keeffe, Jennifer R.; Nishikawa, Hiroyuki; Miyamoto, Keiko; Fox, David; Fukuda, Mamoru; Ohta, Tomohiko; Klevit, Rachel E.

    2003-05-13

    BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RING/RING heterodimer. The BRCA1/BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1/BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn2+-loops and central -helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1/BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.

  18. E3 Ligase Nedd4 Promotes Axon Branching by Downregulating PTEN

    PubMed Central

    Drinjakovic, Jovana; Jung, Hosung; Campbell, Douglas S.; Strochlic, Laure; Dwivedy, Asha; Holt, Christine E.

    2010-01-01

    Summary Regulated protein degradation via the ubiquitin-proteasome system (UPS) plays a central role in building synaptic connections, yet little is known about either which specific UPS components are involved or UPS targets in neurons. We report that inhibiting the UPS in developing Xenopus retinal ganglion cells (RGCs) with a dominant-negative ubiquitin mutant decreases terminal branching in the tectum but does not affect long-range navigation to the tectum. We identify Nedd4 as a prominently expressed E3 ligase in RGC axon growth cones and show that disrupting its function severely inhibits terminal branching. We further demonstrate that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4: not only does Nedd4 regulate PTEN levels in RGC growth cones, but also, the decrease of PTEN rescues the branching defect caused by Nedd4 inhibition. Together our data suggest that Nedd4-regulated PTEN is a key regulator of terminal arborization in vivo. PMID:20159448

  19. Structural Basis for Substrate Selectivity of the E3 Ligase COP1.

    PubMed

    Uljon, Sacha; Xu, Xiang; Durzynska, Izabela; Stein, Sarah; Adelmant, Guillaume; Marto, Jarrod A; Pear, Warren S; Blacklow, Stephen C

    2016-05-01

    COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation. PMID:27041596

  20. Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling.

    PubMed

    Barsi, Julius C; Rajendra, Rashmi; Wu, Jiang I; Artzt, Karen

    2005-10-01

    The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.

  1. Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

    PubMed Central

    Xu, Congfeng; Feng, Kuan; Zhao, Xiaonan; Huang, Shiqian; Cheng, Yiji; Qian, Liu; Wang, Yanan; Sun, Hongxing; Jin, Min; Chuang, Tsung-Hsien; Zhang, Yanyun

    2015-01-01

    Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases. PMID:25484083

  2. Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin.

    PubMed

    Szyk, Agnieszka; Deaconescu, Alexandra M; Piszczek, Grzegorz; Roll-Mecak, Antonina

    2011-11-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms. PMID:22020298

  3. Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

    PubMed

    Ehmann, David E; Demeritt, Julie E; Hull, Kenneth G; Fisher, Stewart L

    2004-05-01

    UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding. PMID:15134649

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

    PubMed Central

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

    2013-01-01

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

  5. Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum.

    PubMed

    Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Cheng, Ai-Xia; Lou, Hong-Xiang

    2015-03-01

    Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.

  6. Activation of Smurf E3 Ligase Promoted by Smoothened Regulates Hedgehog Signaling through Targeting Patched Turnover

    PubMed Central

    Zheng, Xiudeng; Chen, Zhenping; Sun, Liwei; Wang, Hailong; Zhu, Yuanxiang; Zhang, Jing; Yang, Shuyan; Lu, Yi; Sun, Qinmiao; Tao, Yi; Liu, Feng; Zhao, Yun; Chen, Dahua

    2013-01-01

    Hedgehog signaling plays conserved roles in controlling embryonic development; its dysregulation has been implicated in many human diseases including cancers. Hedgehog signaling has an unusual reception system consisting of two transmembrane proteins, Patched receptor and Smoothened signal transducer. Although activation of Smoothened and its downstream signal transduction have been intensively studied, less is known about how Patched receptor is regulated, and particularly how this regulation contributes to appropriate Hedgehog signal transduction. Here we identified a novel role of Smurf E3 ligase in regulating Hedgehog signaling by controlling Patched ubiquitination and turnover. Moreover, we showed that Smurf-mediated Patched ubiquitination depends on Smo activity in wing discs. Mechanistically, we found that Smo interacts with Smurf and promotes it to mediate Patched ubiquitination by targeting the K1261 site in Ptc. The further mathematic modeling analysis reveals that a bidirectional control of activation of Smo involving Smurf and Patched is important for signal-receiving cells to precisely interpret external signals, thereby maintaining Hedgehog signaling reliability. Finally, our data revealed an evolutionarily conserved role of Smurf proteins in controlling Hh signaling by targeting Ptc during development. PMID:24302888

  7. The Evolutionarily Conserved E3 Ubiquitin Ligase AtCHIP Contributes to Plant Immunity

    PubMed Central

    Copeland, Charles; Ao, Kevin; Huang, Yan; Tong, Meixuizi; Li, Xin

    2016-01-01

    Plants possess a sophisticated immune system to recognize and respond to microbial threats in their environment. The level of immune signaling must be tightly regulated so that immune responses can be quickly activated in the presence of pathogens, while avoiding autoimmunity. HSP90s, along with their diverse array of co-chaperones, forms chaperone complexes that have been shown to play both positive and negative roles in regulating the accumulation of immune receptors and regulators. In this study, we examined the role of AtCHIP, an evolutionarily conserved E3 ligase that was known to interact with chaperones including HSP90s in multicellular organisms including fruit fly, Caenorhabditis elegans, plants and human. Atchip knockout mutants display enhanced disease susceptibility to a virulent oomycete pathogen, and overexpression of AtCHIP causes enhanced disease resistance at low temperature. Although CHIP was reported to target HSP90 for ubiquitination and degradation, accumulation of HSP90.3 was not affected in Atchip plants. In addition, protein accumulation of nucleotide-binding, leucine-rich repeat domain immune receptor (NLR) SNC1 is not altered in Atchip mutant. Thus, while AtCHIP plays a role in immunity, it does not seem to regulate the turnover of HSP90 or SNC1. Further investigation is needed in order to determine the exact mechanism behind AtCHIP’s role in regulating plant immune responses. PMID:27014328

  8. Ligase chain reaction to detect Chlamydia trachomatis infection of the cervix.

    PubMed Central

    Schachter, J; Stamm, W E; Quinn, T C; Andrews, W W; Burczak, J D; Lee, H H

    1994-01-01

    We performed a multicenter evaluation of ligase chain reaction (LCR) in the diagnosis of Chlamydia trachomatis infection of the cervix. This LCR provides an amplification of target sequences within the chlamydial cryptic plasmid. The LCR results were compared with those of isolation in cell culture. Discrepant (tissue culture-negative and LCR-positive) test results were resolved by the application of a direct immunofluorescent-antibody test to detect chlamydial elementary bodies and by the use of alternate DNA primers that targeted the chlamydial major outer membrane protein gene. A total of 234 of 2,132 specimens (10.9%) could be confirmed as containing C. trachomatis. Of these, 152 were detected by isolation in cell culture and 221 were detected by LCR. The corresponding sensitivities were 94% for LCR and 65% for cell culture. There was greater variability among study site results for cell culture sensitivity (52 to 92%) than for LCR sensitivity (87 to 98%). The specificity of each test was greater than 99.9%. Thus, LCR offers a highly sensitive nonculture method for detecting chlamydial infection of the cervix. PMID:7814494

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.

    PubMed

    Schmid-Burgk, Jonathan L; Höning, Klara; Ebert, Thomas S; Hornung, Veit

    2016-07-28

    The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications.

  13. Atypical ubiquitin ligase RNF31: the nuclear factor modulator in breast cancer progression.

    PubMed

    Zhu, Jian; Zhuang, Ting; Yang, Huijie; Li, Xin; Liu, Huandi; Wang, Hui

    2016-01-01

    Breast cancer causes the No.1 women cancer prevalence and the No.2 women cancer mortality worldwide. Nuclear receptor/transcriptional factor signaling is aberrant and plays important roles in breast cancer pathogenesis and evolution, such as estrogen receptor α (ERα/ESR1), tumor protein p53 (p53/TP53) and Nuclear factor kappa B (NFκB). About 60-70 % of breast tumors are ERα positive, while approximate 70 % of breast tumors are P53 wild type. Recent studies indicate that nuclear receptors/transcriptional factors could be tightly controlled through protein post-translational modification.The nuclear receptors/transcriptional factors could endure several types of modifications, including phosphorylation, acetylation and ubiquitination. Compared with the other two types of modifications, ubiquitination was mostly linked to protein degradation process, while few researches focused on the functional changes of the target proteins. Until recent years, ubiquitination process is no longer regarded as merely a protein degradation process, but aslo treated as one kind of modification signal.As an atypical E3 ubiquitin ligase, RNF31 was previously found to facilitate NFκB signaling transduction through linear ubiquitination on IKKγ(IκB kinase γ). Our previous studies showed important regulatory functions of RNF31 in controlling important oncogenic pathways in breast cancer, such as ERα and p53. This review highlights recent discoveries on RNF31 functions in nuclear factor modifications, breast cancer progression and possible therapeutic inhibitors targeting RNF31. PMID:27460922

  14. Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

    PubMed

    Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

    2016-08-25

    Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. PMID:27565346

  15. XLF regulates filament architecture of the XRCC4·Ligase IV complex

    PubMed Central

    Hammel, Michal; Yu, Yaping; Fang, Shujuan; Lees-Miller, Susan P.; Tainer, John A.

    2010-01-01

    SUMMARY DNA ligase IV (LigIV) is critical for non-homologous end-joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in human cells, and LigIV activity is regulated by XRCC4 and XLF (XRCC4-like factor) interactions. Here, we employ X-ray scattering (SAXS) data to characterize three-dimensional arrangements in solution for full-length XRCC4, XRCC4 in complex with LigIV tandem BRCT domains and XLF, plus the XRCC4·XLF·BRCT2 complex. XRCC4 forms tetramers mediated through a head-to-head interface and the XRCC4 C-terminal coiled-coil region folds back on itself to support this interaction. The interaction between XLF and XRCC4 is also mediated via head-to-head interactions. In the XLF·XRCC4·BRCT complex, alternating repeating units of XLF and XRCC4·BRCT place the BRCT domain on one side of the filament. Collective results identify XRCC4 and XLF filaments suitable to align DNA molecules and function to facilitate LigIV end joining required for DSB repair in vivo. PMID:21070942

  16. Structural Basis of Dimerization-dependent Ubiquitination by the SCFFbx4 Ubiquitin Ligase

    SciTech Connect

    Li, Y.; Hao, B

    2010-01-01

    The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F-box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF{sup Fbx4} (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adopts a compact {alpha}/{beta} fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

  17. Fine Tuning of the UPR by the Ubiquitin Ligases Siah1/2

    PubMed Central

    Scortegagna, Marzia; Kim, Hyungsoo; Li, Jian-Liang; Yao, Hang; Brill, Laurence M.; Han, Jaeseok; Lau, Eric; Bowtell, David; Haddad, Gabriel; Kaufman, Randal J.; Ronai, Ze'ev A.

    2014-01-01

    The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). Yet, it is not known how UPR-signaling coordinates adaptation versus cell death. Previous studies suggested that signaling through PERK/ATF4 is required for cell death. We show that high levels of ER stress (i.e., ischemia-like conditions) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1. In turn, Siah1/2 attenuates proline hydroxylation of ATF4, resulting in its stabilization, thereby augmenting ER stress output. Conversely, ATF4 activation is reduced upon Siah1/2 KD in cultured cells, which attenuates ER stress-induced cell death. Notably, Siah1a+/−::Siah2−/− mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death, compared with the wild type (WT) mice. In all, Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions. PMID:24809345

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

    PubMed Central

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

    2016-01-01

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

  19. Structure guided design of biotin protein ligase inhibitors for antibiotic discovery.

    PubMed

    Paparella, Ashleigh S; Soares da Costa, Tatiana P; Yap, Min Y; Tieu, William; Wilce, Matthew C J; Booker, Grant W; Abell, Andrew D; Polyak, Steven W

    2014-01-01

    Biotin protein ligase (BPL) represents a promising target for the discovery of new antibacterial chemotherapeutics. Here we review the central role of BPL for the survival and virulence of clinically important Staphylococcus aureus in support of this claim. X-ray crystallography structures of BPLs in complex with ligands and small molecule inhibitors provide new insights into the mechanism of protein biotinylation, and a template for structure guided approaches to the design of inhibitors for antibacterial discovery. Most BPLs employ an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5´-AMP from substrates biotin and ATP. Recent studies reporting chemical analogs of biotin and biotinyl-5´-AMP as BPL inhibitors that represent new classes of anti-S. aureus agents are reviewed. We highlight strategies to selectively inhibit bacterial BPL over the mammalian equivalent using a 1,2,3-triazole isostere to replace the labile phosphoanhydride naturally present in biotinyl-5´-AMP. A novel in situ approach to improve the detection of triazole-based inhibitors is also presented that could potentially be widely applied to other protein targets.

  20. A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen*

    PubMed Central

    Sheng, Yi; Hong, Jenny H.; Doherty, Ryan; Srikumar, Tharan; Shloush, Jonathan; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Neculai, Dante; Wan, Janet W.; Kim, Sung K.; Arrowsmith, Cheryl H.; Raught, Brian; Dhe-Paganon, Sirano

    2012-01-01

    Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. PMID:22496338

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

    PubMed Central

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

    2015-01-01

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

  2. Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling.

    PubMed

    Polekhina, Galina; House, Colin M; Traficante, Nadia; Mackay, Joel P; Relaix, Frédéric; Sassoon, David A; Parker, Michael W; Bowtell, David D L

    2002-01-01

    Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 A resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded beta-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-alpha receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-alpha-mediated NF-kappa B activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.

  3. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.

    PubMed

    Schmid-Burgk, Jonathan L; Höning, Klara; Ebert, Thomas S; Hornung, Veit

    2016-01-01

    The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications. PMID:27465542

  4. Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

    PubMed Central

    Petrelli, Riccardo; De la Mora-Rey, Teresa; Tiwari, Divya; Liu, Feng; Dawadi, Surrendra; Nandakumar, Madhumitha; Rhee, Kyu Y.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) responsible for both latent and symptomatic tuberculosis (TB) remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KD's below 2 nM. Additionally, we obtained high-resolution co-crystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhanced accumulation of a C-2′-α analog over the corresponding C-2′-β analog, consistent with their differential whole-cell activity. PMID:26299766

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

    PubMed Central

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

    2016-01-01

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

  6. Transcription factor RFX1 is ubiquitinated by E3 ligase STUB1 in systemic lupus erythematosus.

    PubMed

    Guo, Yu; Zhao, Ming; Lu, Qianjin

    2016-08-01

    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by complex interactions between genes and the environment. The expression level of transcription factor regulatory factor X 1 (RFX1) is reduced in T cells from SLE patients. RFX1 can regulate epigenetic modifications of CD70 and CD11a and plays an important role in the development of SLE. However, the mechanisms that mediate reduction of RFX1 in SLE are unclear. Here, we demonstrate that RFX1 protein expression can be tightly regulated by polyubiquitination-mediated proteosomal degradation via STIP1 homology and U-box containing protein 1 (STUB1). The E3 ligase STUB1 is upregulated in CD4(+)T cells of SLE patients compared to healthy subjects. Overexpression of STUB1 in CD4(+)T cells leads to upregulation of levels of CD70 and CD11a in T cells. The modulation of STUB1 activity may provide a novel therapeutic approach for SLE. PMID:27283392

  7. Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase.

    PubMed

    Li, Zhi; Nair, Satish K

    2015-11-01

    Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) and ATP, with AMP and coenzyme A (CoA), and with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP. The Nt4CL2-Mg(2+)-ATP structure is captured in the adenylate-forming conformation, whereas the other structures are in the thioester-forming conformation. These structures represent a rare example of an ANL enzyme visualized in both conformations, and also reveal the binding determinants for both CoA and the hydroxycinnamate substrate. Kinetic studies of structure-based variants were used to identify residues crucial to catalysis, ATP binding, and hydroxycinnamate specificity. Lastly, we characterize a deletion mutant of Nt4CL2 that possesses the unusual sinapinate-utilizing activity. These studies establish a molecular framework for the engineering of this versatile biocatalyst. PMID:26412334

  8. Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases.

    PubMed

    Broemer, Meike; Tenev, Tencho; Rigbolt, Kristoffer T G; Hempel, Sophie; Blagoev, Blagoy; Silke, John; Ditzel, Mark; Meier, Pascal

    2010-12-10

    The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.

  9. The Effect of Cytidine on the Structure and Function of an RNA Ligase Ribozyme

    NASA Technical Reports Server (NTRS)

    Rogers, Jeff; Joyce, Gerald F.

    2001-01-01

    A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random- sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5' -phosphodiester linkage with a catalytic rate of 0.016/min. The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32/min. The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

  10. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  11. Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum.

    PubMed

    Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Cheng, Ai-Xia; Lou, Hong-Xiang

    2015-03-01

    Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate. PMID:25593011

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

    PubMed Central

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

    2015-01-01

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

  13. New role of E3 ubiquitin ligase in the regulation of necroptosis

    PubMed Central

    Seo, Jinho; Lee, Eun-Woo; Song, Jaewhan

    2016-01-01

    Necroptosis is a well-known form of caspase-independent cell death. Necroptosis can be triggered by various extrinsic stimuli, including death ligands in the presence of receptorinteracting protein kinase 3 (RIPK3), a key mediator of necroptosis induction. Our recent studies have revealed that C-terminus HSC-70 interacting protein (CHIP), an E3 ligase, can function as an inhibitor of necroptosis. CHIP−/− mouse embryonic fibroblast showed higher sensitivity to necrotic stimuli than wild-type mouse embryonic fibroblast cells. Deleterious effects of CHIP knockout MEFs were retrieved by RIPK3 depletion. We found that CHIP negatively regulated RIPK3 and RIPK1 by ubiquitylation- and lysosome- dependent degradation. In addition, CHIP−/− mice showed postnatal lethality with intestinal defects that could be rescued by crossing with RIPK3−/− mice. These results suggest that CHIP is a negative regulator of RIPK1 and RIPK3, thus inhibiting necroptosis. [BMB Reports 2016; 49(5): 247-248] PMID:27099235

  14. The -4 phenylalanine is required for substrate ubiquitination catalyzed by HECT ubiquitin ligases.

    PubMed

    Salvat, Catherine; Wang, Guangli; Dastur, Anahita; Lyon, Nancy; Huibregtse, Jon M

    2004-04-30

    The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.

  15. Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2010-04-30

    Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R = 19.9%, R(free) = 25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site and disrupts the previously observed Mg(2+) coordination in the ATP-binding site. l-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R = 18.1%, R(free) = 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

  16. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

    PubMed Central

    Kumar, Atul; Aguirre, Jacob D; Condos, Tara EC; Martinez-Torres, R Julio; Chaugule, Viduth K; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Mercier, Pascal; Knebel, Axel; Spratt, Donald E; Barber, Kathryn R; Shaw, Gary S; Walden, Helen

    2015-01-01

    The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin. PMID:26254304

  17. Regulation of a Spindle Positioning Factor at Kinetochores by SUMO-Targeted Ubiquitin Ligases.

    PubMed

    Schweiggert, Jörg; Stevermann, Lea; Panigada, Davide; Kammerer, Daniel; Liakopoulos, Dimitris

    2016-02-22

    Correct function of the mitotic spindle requires balanced interplay of kinetochore and astral microtubules that mediate chromosome segregation and spindle positioning, respectively. Errors therein can cause severe defects ranging from aneuploidy to developmental disorders. Here, we describe a protein degradation pathway that functionally links astral microtubules to kinetochores via regulation of a microtubule-associated factor. We show that the yeast spindle positioning protein Kar9 localizes not only to astral but also to kinetochore microtubules, where it becomes targeted for proteasomal degradation by the SUMO-targeted ubiquitin ligases (STUbLs) Slx5-Slx8. Intriguingly, this process does not depend on preceding sumoylation of Kar9 but rather requires SUMO-dependent recruitment of STUbLs to kinetochores. Failure to degrade Kar9 leads to defects in both chromosome segregation and spindle positioning. We propose that kinetochores serve as platforms to recruit STUbLs in a SUMO-dependent manner in order to ensure correct spindle function by regulating levels of microtubule-associated proteins. PMID:26906737

  18. Proteomic analysis and identification of cellular interactors of the giant ubiquitin ligase HERC2.

    PubMed

    Galligan, Jeffrey T; Martinez-Noël, Gustavo; Arndt, Verena; Hayes, Sebastian; Chittenden, Thomas W; Harper, J Wade; Howley, Peter M

    2015-02-01

    HERC2 is a large E3 ubiquitin ligase with multiple structural domains that has been implicated in an array of cellular processes. Mutations in HERC2 are linked to developmental delays and impairment caused by nervous system dysfunction, such as Angelman Syndrome and autism-spectrum disorders. However, HERC2 cellular activity and regulation remain poorly understood. We used a broad proteomic approach to survey the landscape of cellular proteins that interact with HERC2. We identified nearly 300 potential interactors, a subset of which we validated binding to HERC2. The potential HERC2 interactors included the eukaryotic translation initiation factor 3 complex, the intracellular transport COPI coatomer complex, the glycogen regulator phosphorylase kinase, beta-catenin, PI3 kinase, and proteins involved in fatty acid transport and iron homeostasis. Through a complex bioinformatic analysis of potential interactors, we linked HERC2 to cellular processes including intracellular protein trafficking and transport, metabolism of cellular energy, and protein translation. Given its size, multidomain structure, and association with various cellular activities, HERC2 may function as a scaffold to integrate protein complexes and bridge critical cellular pathways. This work provides a significant resource with which to interrogate HERC2 function more deeply and evaluate its contributions to mechanisms governing cellular homeostasis and disease. PMID:25476789

  19. Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

    SciTech Connect

    Duda,D.; Borg, L.; Scott, D.; Hunt, H.; Hammel, M.; Schulman, B.

    2008-01-01

    Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8{approx}Cul5ctd-Rbx1 and SAXS analysis of NEDD8{approx}Cul1ctd-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.

  20. The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron

    PubMed Central

    Habeck, Gregor; Ebner, Felix A.; Shimada-Kreft, Hiroko

    2015-01-01

    Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates. PMID:25918226

  1. Proteomic insight into the effects of the Salmonella ubiquitin ligase SlrP on host cells.

    PubMed

    Cordero-Alba, Mar; García-Gómez, Juan José; Aguilera-Herce, Julia; Ramos-Morales, Francisco

    2016-04-01

    The virulence of the human and animal pathogen Salmonella enterica serovar Typhimurium is dependent on two type III secretion systems. These systems translocate proteins called effectors into eukaryotic host cells. SlrP is a Salmonella type III secretion effector with ubiquitin ligase activity. Here, we used two complementary proteomic approaches, two-dimensional gel electrophoresis and iTRAQ (isobaric tags for relative and absolute quantification) to study the consequences of the presence of SlrP in human epithelial cells. We identified 37 proteins that were differentially expressed in HeLa cells expressing slrP compared to control cells. Microarray analysis revealed that more than a half of differentially expressed proteins did not show changes in the transcriptome, suggesting post-transcriptional regulation. A gene ontology overrepresentation test carried out on the differentially expressed proteins revealed enrichment of ontology terms related to several types of junctions mediating adhesion in epithelial cells. Consistently, slrP-transfected cells showed defects in migration and adhesion. Our results suggest that the modification of cell-cell interaction ability of the host could be one of the final consequences of the action of SlrP during an infection.

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

    SciTech Connect

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

    2010-12-01

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

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

    SciTech Connect

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

    2010-09-17

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

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

    PubMed Central

    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

    2013-01-01

    Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), 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 specific recognition of poly-SUMO2/3 chains, and used Ubc13–Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response. PMID:23751493

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

    PubMed Central

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

    2014-01-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator. PMID:24413661

  6. Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination.

    PubMed

    Xu, Congfeng; Feng, Kuan; Zhao, Xiaonan; Huang, Shiqian; Cheng, Yiji; Qian, Liu; Wang, Yanan; Sun, Hongxing; Jin, Min; Chuang, Tsung-Hsien; Zhang, Yanyun

    2014-01-01

    Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.

  7. E3 ubiquitin ligase RNF31 cooperates with DAX-1 in transcriptional repression of steroidogenesis.

    PubMed

    Ehrlund, Anna; Anthonisen, Elin Holter; Gustafsson, Nina; Venteclef, Nicolas; Robertson Remen, Kirsten; Damdimopoulos, Anastasios E; Galeeva, Anastasia; Pelto-Huikko, Markku; Lalli, Enzo; Steffensen, Knut R; Gustafsson, Jan-Ake; Treuter, Eckardt

    2009-04-01

    Genetic and experimental evidence points to a critical involvement of the atypical mammalian orphan receptor DAX-1 in reproductive development and steroidogenesis. Unlike conventional nuclear receptors, DAX-1 appears not to function as a DNA-bound transcription factor. Instead, it has acquired the capability to act as a transcriptional corepressor of steroidogenic factor 1 (SF-1). The interplay of DAX-1 and SF-1 is considered a central, presumably ligand-independent element of adrenogonadal development and function that requires tight regulation. This raises a substantial interest in identifying its modulators and the regulatory signals involved. Here, we uncover molecular mechanisms that link DAX-1 to the ubiquitin modification system via functional interaction with the E3 ubiquitin ligase RNF31. We demonstrate that RNF31 is coexpressed with DAX-1 in steroidogenic tissues and participates in repressing steroidogenic gene expression. We provide evidence for the in vivo existence of a corepressor complex containing RNF31 and DAX-1 at the promoters of the StAR and CYP19 genes. Our data suggest that RNF31 functions to stabilize DAX-1, which might be linked to DAX-1 monoubiquitination. In conclusion, RNF31 appears to be required for DAX-1 to repress transcription, provides means to regulate DAX-1 in ligand-independent ways, and emerges as a relevant coregulator of steroidogenic pathways governing physiology and disease. PMID:19237537

  8. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

    PubMed Central

    Schmid-Burgk, Jonathan L.; Höning, Klara; Ebert, Thomas S.; Hornung, Veit

    2016-01-01

    The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications. PMID:27465542

  9. Mms21 SUMO Ligase Activity Promotes Nucleolar Function in Saccharomyces cerevisiae

    PubMed Central

    Kim, Dong-Hwan; Harris, Bethany; Wang, Fei; Seidel, Chris; McCroskey, Scott; Gerton, Jennifer L.

    2016-01-01

    The budding yeast E3 SUMO ligase Mms21, also known as Nse2, is a component of the Smc5/6 complex, which regulates sister chromatid cohesion, DNA replication, and repair. Our study shows that the mms21RINGΔ mutant exhibits (1) reduced ribosomal RNA production; (2) nuclear accumulation of ribosomal proteins; (3) elevated Gcn4 translation, indicating translational stress; and (4) upregulation of Gcn4 targets. Genes involved in ribosome biogenesis and translation are downregulated in the mms21RINGΔ mutant. We identified RPL19A as a novel genetic suppressor of the mms21RINGΔ mutant. Deletion of RPL19A partially suppresses growth defects in both smc5-6 and mms21RINGΔ mutants as well as nuclear accumulation of ribosome subunits in the mms21RINGΔ mutant. Deletion of a previously identified strong suppressor, MPH1, rescues both the accumulation of ribosome subunits and translational stress. This study suggests that the Smc5/6 complex supports nucleolar function. PMID:27510371

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

    PubMed

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

    2013-08-01

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

  11. Dyrk1A phosphorylates parkin at Ser-131 and negatively regulates its ubiquitin E3 ligase activity.

    PubMed

    Im, Eunju; Chung, Kwang Chul

    2015-08-01

    Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady-state levels through the ubiquitin-proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co-localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser-131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A-mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin-conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A-mediated phosphorylation inhibited the neuroprotective action of parkin against 6-hydroxydopamine toxicity in dopaminergic SH-SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD-inducing pathological conditions. Mutations of parkin are linked to autosomal recessive forms of familial Parkinson's disease (PD). According to its functional relevance in abnormal protein aggregation and neuronal cell death, a number of post-translational modifications regulate the ubiquitin E3 ligase activity of parkin. Here we propose a novel inhibitory mechanism of parkin E3 ubiquitin ligase through dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A)-mediated phosphorylation as well as its neuroprotective action against 6-hydroxydopamine (6-OHDA)-induced cell death

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

    PubMed Central

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

    2014-01-01

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

  13. E3 SUMO ligase AtSIZ1 positively regulates SLY1-mediated GA signalling and plant development.

    PubMed

    Kim, Sung-Il; Park, Bong Soo; Kim, Do Youn; Yeu, Song Yion; Song, Sang Ik; Song, Jong Tae; Seo, Hak Soo

    2015-07-15

    Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.

  14. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    PubMed

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients.

  15. Differential gene expression of muscle-specific ubiquitin ligase MAFbx/Atrogin-1 and MuRF1 in response to immobilization-induced atrophy of slow-twitch and fast-twitch muscles.

    PubMed

    Okamoto, Takeshi; Torii, Suguru; Machida, Shuichi

    2011-11-01

    We examined muscle-specific ubiquitin ligases MAFbx/Atrogin-1 and MuRF1 gene expression resulting from immobilization-induced skeletal muscle atrophy of slow-twitch soleus and fast-twitch plantaris muscles. Male C57BL/6 mice were subjected to hindlimb immobilization, which induced similar percentage decreases in muscle mass in the soleus and plantaris muscles. Expression of MAFbx/Atrogin-1 and MuRF1 was significantly greater in the plantaris muscle than in the soleus muscle during the early stage of atrophy. After a 3-day period of atrophy, total FOXO3a protein level had increased in both muscles, while phosphorylated FOXO3a protein had decreased in the plantaris muscle, but not in the soleus muscle. PGC-1α protein expression did not change following immobilization in both muscles, but basal PGC-1α protein in the soleus was markedly higher than that in plantaris muscles. These data suggest that although soleus and plantaris muscles atrophied to a similar extent and that muscle-specific ubiquitin protein ligases (E3) may contribute more to the atrophy of fast-twitch muscle than to that of slow-twitch muscle during immobilization.

  16. The E3 Ubiquitin Ligase ARIH1 Protects against Genotoxic Stress by Initiating a 4EHP-Mediated mRNA Translation Arrest

    PubMed Central

    von Stechow, Louise; Typas, Dimitris; Carreras Puigvert, Jordi; Oort, Laurens; Siddappa, Ramakrishnaiah; Pines, Alex; Vrieling, Harry; van de Water, Bob

    2015-01-01

    DNA damage response signaling is crucial for genome maintenance in all organisms and is corrupted in cancer. In an RNA interference (RNAi) screen for (de)ubiquitinases and sumoylases modulating the apoptotic response of embryonic stem (ES) cells to DNA damage, we identified the E3 ubiquitin ligase/ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein abundance increased after DNA damage through attenuation of proteasomal degradation that required ATM signaling. Accumulated ARIH1 associated with 4EHP, and in turn, this competitive inhibitor of the eukaryotic translation initiation factor 4E (eIF4E) underwent increased nondegradative ubiquitination upon DNA damage. Genotoxic stress led to an enrichment of ARIH1 in perinuclear, ribosome-containing regions and triggered 4EHP association with the mRNA 5′ cap as well as mRNA translation arrest in an ARIH1-dependent manner. Finally, restoration of DNA damage-induced translation arrest in ARIH1-depleted cells by means of an eIF2 inhibitor was sufficient to reinstate resistance to genotoxic stress. These findings identify ARIH1 as a potent mediator of DNA damage-induced translation arrest that protects stem and cancer cells against genotoxic stress. PMID:25624349

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

    PubMed Central

    Vandewalle, Joke; Langen, Marion; Zschaetzsch, Marlen; Nijhof, Bonnie; Kramer, Jamie M.; Brems, Hilde; Bauters, Marijke; Lauwers, Elsa; Srahna, Mohammed; Marynen, Peter; Verstreken, Patrik; Schenck, Annette; Hassan, Bassem A.; Froyen, Guy

    2013-01-01

    We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of β-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/β-catenin pathway possibly by enhancing the degradation of dsh. PMID:24303071

  18. Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/β-catenin pathway in a Drosophila model for intellectual disability.

    PubMed

    Vandewalle, Joke; Langen, Marion; Zschätzsch, Marlen; Zschaetzsch, Marlen; Nijhof, Bonnie; Kramer, Jamie M; Brems, Hilde; Bauters, Marijke; Lauwers, Elsa; Srahna, Mohammed; Marynen, Peter; Verstreken, Patrik; Schenck, Annette; Hassan, Bassem A; Froyen, Guy

    2013-01-01

    We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of β-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/β-catenin pathway possibly by enhancing the degradation of dsh.

  19. Highly sensitive DNA detection and point mutation identification: an electrochemical approach based on the combined use of ligase and reverse molecular beacon.

    PubMed

    Wu, Zai-Sheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2007-06-01

    A novel strategy is described for highly sensitive DNA detection and point mutation identification based on the combination of reverse molecular beacon with DNA ligase. A 5'-phosphoryl and 3'-ferrocene terminated DNA sequence is used as detection probe, which may be ligated to capture DNA immobilized on an electrode surface in the presence of a target DNA strand that is complementary to the ends of each DNA, since this allows formation of a nicked, double-stranded DNA. The ligation product may form a hairpin structure after the removal of target DNA. By this method, target DNA can be determined in the range from 3.4 x 10(-12) to 1.4 x 10(-7) M with a detection limit of 1.0 x 10(-12) M. In contrast to existing methods based on the conformation change of redox-labeled oligonucleotides, the proposed strategy offers several substantial advantages: first, the background peak current is eliminated as the ferrocene (Fc)-tagged oligonucleotide probe is specifically ligated to capture DNA; second, a "signal-on" mechanism makes the current intensity increase with increasing target DNA concentration; third, improved current signal is obtained due to the formation of the hairpin structure of ligation products. Additionally, the present system exhibits excellent capability to discriminate mutant target sequences from fully complementary target sequences.

  20. Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae.

    PubMed

    Rai, Ragini; Varma, Satya P M V; Shinde, Nikhil; Ghosh, Shilpa; Kumaran, Srikala P; Skariah, Geena; Laloraya, Shikha

    2011-04-22

    The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast. PMID:21324902

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

    PubMed

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

    2013-10-11

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

  2. Discovery of the first inhibitors of bacterial enzyme D-aspartate ligase from Enterococcus faecium (Aslfm).

    PubMed

    Škedelj, Veronika; Perdih, Andrej; Brvar, Matjaž; Kroflič, Ana; Dubbée, Vincent; Savage, Victoria; O'Neill, Alex J; Solmajer, Tom; Bešter-Rogač, Marija; Blanot, Didier; Hugonnet, Jean-Emmanuel; Magnet, Sophie; Arthur, Michel; Mainardi, Jean-Luc; Stojan, Jure; Zega, Anamarija

    2013-09-01

    The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.

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

    PubMed Central

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

    2016-01-01

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

  4. Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry

    PubMed Central

    Yu, Jian; Cai, Yu-Xiang; Zhu, Pan-Pan; Liu, Chang-Ying; Zhao, Ai-Chun; Lü, Rui-Hua; Li, Meng-Jiao; Xu, Feng-Xiang; Yu, Mao-De

    2016-01-01

    A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol. PMID:27213624

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

    The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use. PMID:26392552

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

    PubMed

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

    2015-10-01

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

  8. An impaired ubiquitin ligase complex favors initial growth of auxotrophic yeast strains in synthetic grape must.

    PubMed

    Mangado, Ana; Tronchoni, Jordi; Morales, Pilar; Novo, Maite; Quirós, Manuel; Gonzalez, Ramon

    2015-02-01

    We used experimental evolution in order to identify genes involved in the adaptation of Saccharomyces cerevisiae to the early stages of alcoholic fermentation. Evolution experiments were run for about 200 generations, in continuous culture conditions emulating the initial stages of wine fermentation. We performed whole-genome sequencing of four adapted strains from three independent evolution experiments. Mutations identified in these strains pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor specifically involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. While a loss-of-function mutation in BUL1 seems to be enough to confer a selective advantage under these assay conditions, this did not seem to be the case for RSP5 mutated strains, which required additional mutations, probably compensating for the detrimental effect of altered Rsp5p activity on essential cellular functions. The power of this experimental approach is illustrated by the identification of four independent mutants, each with a limited number of SNPs, affected within the same pathway. However, in order to obtain information relevant for a specific biotechnological process, caution must be taken in the choice of the background yeast genotype (as shown in this case for auxotrophies). In addition, the use of very stable continuous fermentation conditions might lead to the selection of a rather limited number of adaptive responses that would mask other possible targets for genetic improvement. PMID:25620600

  9. Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

    PubMed

    Desrochers, Guillaume; Lussier-Price, Mathieu; Omichinski, James G; Angers, Annie

    2015-12-22

    Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested. PMID:26613292

  10. Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mul-berry.

    PubMed

    Wang, Chuan-Hong; Yu, Jian; Cai, Yu-Xiang; Zhu, Pan-Pan; Liu, Chang-Ying; Zhao, Ai-Chun; Lü, Rui-Hua; Li, Meng-Jiao; Xu, Feng-Xiang; Yu, Mao-De

    2016-01-01

    A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol. PMID:27213624

  11. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase.

    PubMed

    Bauer, Robert J; Evans, Thomas C; Lohman, Gregory J S

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

  12. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    PubMed Central

    Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

  13. Homozygous DNA ligase IV R278H mutation in mice leads to leaky SCID and represents a model for human LIG4 syndrome

    PubMed Central

    Rucci, Francesca; Notarangelo, Luigi D.; Fazeli, Alex; Patrizi, Laura; Hickernell, Thomas; Paganini, Tiziana; Coakley, Kristen M.; Detre, Cynthia; Keszei, Marton; Walter, Jolan E.; Feldman, Lauren; Cheng, Hwei-Ling; Poliani, Pietro Luigi; Wang, Jing H.; Balter, Barbara B.; Recher, Mike; Andersson, Emma-Maria; Zha, Shan; Giliani, Silvia; Terhorst, Cox; Alt, Frederick W.; Yan, Catherine T.

    2010-01-01

    DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4R278H/R278H (Lig4R/R) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4R/R mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans. PMID:20133615

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

    PubMed

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

    2008-02-01

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

  15. Submicroscopic Duplications of the Hydroxysteroid Dehydrogenase HSD17B10 and the E3 Ubiquitin Ligase HUWE1 Are Associated with Mental Retardation

    PubMed Central

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

    2008-01-01

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too. PMID:18252223

  16. E3 Ubiquitin Ligase, WWP1, Interacts with AMPKα2 and Down-regulates Its Expression in Skeletal Muscle C2C12 Cells*

    PubMed Central

    Lee, Jung Ok; Lee, Soo Kyung; Kim, Nami; Kim, Ji Hae; You, Ga Young; Moon, Ji Wook; Jie, Sha; Kim, Su Jin; Lee, Yong Woo; Kang, Ho Jin; Lim, Yongchul; Park, Sun Hwa; Kim, Hyeon Soo

    2013-01-01

    It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway. PMID:23293026

  17. The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple substrate proteins and its heterogeneous overexpression enhances acquired thermotolerance

    PubMed Central

    Lim, Sung Don; Cho, Hyun Yong; Park, Yong Chan; Ham, Deok Jae; Lee, Ju Kyong; Jang, Cheol Seong

    2013-01-01

    Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear–cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock. PMID:23698632

  18. Skp1-Cullin-F-box (SCF)-type ubiquitin ligase FBXW7 negatively regulates spermatogonial stem cell self-renewal

    PubMed Central

    Kanatsu-Shinohara, Mito; Onoyama, Ichiro; Nakayama, Keiichi I.; Shinohara, Takashi

    2014-01-01

    Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box–type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC. PMID:24879440

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

    PubMed

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

    2016-10-01

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

  20. Mutation in a D-alanine-D-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress.

    PubMed

    Jofré, Edgardo; Fischer, Sonia; Príncipe, Analía; Castro, Marina; Ferrari, Walter; Lagares, Antonio; Mori, Gladys

    2009-01-01

    Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.

  1. The E3 ligase APC/C-Cdh1 is required for associative fear memory and long-term potentiation in the amygdala of adult mice.

    PubMed

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

    2012-12-14

    The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating knockout mice where Cdh1 was conditionally eliminated from the forebrain post-developmentally. Although spatial learning and memory in the Morris water maze (MWM) was normal, the Cdh1 conditional knockout (cKO) mice displayed enhanced reversal learning in the MWM and in a water-based Y maze. In addition, we found that the Cdh1 cKO mice had impaired associative fear memory and exhibited impaired long-term potentiation (LTP) in amygdala slices. Finally, we observed increased expression of Shank1 and NR2A expression in amygdalar slices from the Cdh1 cKO mice following the induction of LTP, suggesting a possible molecular mechanism underlying the behavioral and synaptic plasticity impairments displayed in these mice. Our findings are consistent with a role for the APC/C-Cdh1 in fear memory and synaptic plasticity in the amygdala.

  2. The Mid2 X-linked Intellectual Disability Ubiquitin Ligase Associates with Astrin and Regulates Astrin Levels to Promote Cell Division

    PubMed Central

    Gholkar, Ankur A.; Senese, Silvia; Lo, Yu-Chen; Vides, Edmundo; Contreras, Ely; Hodara, Emmanuelle; Capri, Joseph; Whitelegge, Julian P.; Torres, Jorge Z.

    2015-01-01

    SUMMARY Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that Astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409 and is degraded during cytokinesis. Mid2 depletion led to Astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant Astrin in Astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of Astrin on K409, which is critical for its degradation and proper cytokinesis. These results may help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities. PMID:26748699

  3. The X-Linked-Intellectual-Disability-Associated Ubiquitin Ligase Mid2 Interacts with Astrin and Regulates Astrin Levels to Promote Cell Division.

    PubMed

    Gholkar, Ankur A; Senese, Silvia; Lo, Yu-Chen; Vides, Edmundo; Contreras, Ely; Hodara, Emmanuelle; Capri, Joseph; Whitelegge, Julian P; Torres, Jorge Z

    2016-01-12

    Mid1 and Mid2 are ubiquitin ligases that regulate microtubule dynamics and whose mutation is associated with X-linked developmental disorders. We show that astrin, a microtubule-organizing protein, co-purifies with Mid1 and Mid2, has an overlapping localization with Mid1 and Mid2 at intercellular bridge microtubules, is ubiquitinated by Mid2 on lysine 409, and is degraded during cytokinesis. Mid2 depletion led to astrin stabilization during cytokinesis, cytokinetic defects, multinucleated cells, and cell death. Similarly, expression of a K409A mutant astrin in astrin-depleted cells led to the accumulation of K409A on intercellular bridge microtubules and an increase in cytokinetic defects, multinucleated cells, and cell death. These results indicate that Mid2 regulates cell division through the ubiquitination of astrin on K409, which is critical for its degradation and proper cytokinesis. These results could help explain how mutation of MID2 leads to misregulation of microtubule organization and the downstream disease pathology associated with X-linked intellectual disabilities.

  4. C-terminal region of bacterial Ku controls DNA bridging, DNA threading and recruitment of DNA ligase D for double strand breaks repair

    PubMed Central

    McGovern, Stephen; Baconnais, Sonia; Roblin, Pierre; Nicolas, Pierre; Drevet, Pascal; Simonson, Héloïse; Piétrement, Olivier; Charbonnier, Jean-Baptiste; Le Cam, Eric; Noirot, Philippe; Lecointe, François

    2016-01-01

    Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region. PMID:26961308

  5. Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue, Shaggy

    PubMed Central

    Moncrieff, Sophie; Moncan, Matthieu; Scialpi, Flavia; Ditzel, Mark

    2015-01-01

    Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals. PMID:26334301

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

    PubMed

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

    2016-04-01

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

  7. Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

    PubMed

    Shrivastava, Nidhi; Nag, Jeetendra Kumar; Misra-Bhattacharya, Shailja

    2012-01-01

    The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies. PMID:22815933

  8. Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase*

    PubMed Central

    Edwards, Daniel J.; Streich, Frederick C.; Ronchi, Virginia P.; Todaro, Dustin R.; Haas, Arthur L.

    2014-01-01

    The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of 125I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼125I-ubiquitin thioester co-substrate in the nanomolar range ([S]½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼125I-ubiquitin thioester (Ki = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (Ki = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the kcat but not [S]½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe395. Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349–10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases. PMID:25342744

  9. The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase.

    PubMed

    Pawlowska, Roza; Korczynski, Dariusz; Nawrot, Barbara; Stec, Wojciech J; Chworos, Arkadiusz

    2016-08-01

    T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors.

  10. Arabidopsis PIAL1 and 2 Promote SUMO Chain Formation as E4-Type SUMO Ligases and Are Involved in Stress Responses and Sulfur Metabolism[C][W][OPEN

    PubMed Central

    Tomanov, Konstantin; Zeschmann, Anja; Hermkes, Rebecca; Eifler, Karolin; Ziba, Ionida; Grieco, Michele; Novatchkova, Maria; Hofmann, Kay; Hesse, Holger; Bachmair, Andreas

    2014-01-01

    The Arabidopsis thaliana genes PROTEIN INHIBITOR OF ACTIVATED STAT LIKE1 (PIAL1) and PIAL2 encode proteins with SP-RING domains, which occur in many ligases of the small ubiquitin-related modifier (SUMO) conjugation pathway. We show that PIAL1 and PIAL2 function as SUMO ligases capable of SUMO chain formation and require the SUMO-modified SUMO-conjugating enzyme SCE1 for optimal activity. Mutant analysis indicates a role for PIAL1 and 2 in salt stress and osmotic stress responses, whereas under standard conditions, the mutants show close to normal growth. Mutations in PIAL1 and 2 also lead to altered sulfur metabolism. We propose that, together with SUMO chain binding ubiquitin ligases, these enzymes establish a pathway for proteolytic removal of sumoylation substrates. PMID:25415977

  11. Enhanced waterlogging tolerance in barley by manipulation of expression of the N-end rule pathway E3 ligase PROTEOLYSIS6.

    PubMed

    Mendiondo, Guillermina M; Gibbs, Daniel J; Szurman-Zubrzycka, Miriam; Korn, Arnd; Marquez, Julietta; Szarejko, Iwona; Maluszynski, Miroslaw; King, John; Axcell, Barry; Smart, Katherine; Corbineau, Francoise; Holdsworth, Michael J

    2016-01-01

    Increased tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh., the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII ethylene response factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-cysteine (Cys). Here, we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore, we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygen-sensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging, transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll, and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions IN Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals. PMID:25657015

  12. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  13. Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD)

    PubMed Central

    Dutta, Kaushik; Natarajan, Aswin; Nair, Pravin A.; Shuman, Stewart; Ghose, Ranajeet

    2014-01-01

    DNA ligase D (LigD), consisting of polymerase, ligase and phosphoesterase domains, is the essential catalyst of the bacterial non-homologous end-joining pathway of DNA double-strand break repair. The phosphoesterase (PE) module performs manganese-dependent 3’-phosphomonoesterase and 3’-ribonucleoside resection reactions that heal broken ends in preparation for sealing. LigD PE exemplifies a structurally and mechanistically unique class of DNA end-processing enzymes. Here, we present the resonance assignments of the PE domain of Pseudomonas aeruginosa LigD comprising the N-terminal 177 residues. PMID:21213076

  14. Lack of Cul4b, an E3 Ubiquitin Ligase Component, Leads to Embryonic Lethality and Abnormal Placental Development

    PubMed Central

    Yuan, Jupeng; Qian, Yanyan; Sun, Wenjie; Zou, Yongxin; Guo, Chenhong; Chen, Bingxi; Shao, Changshun; Gong, Yaoqin

    2012-01-01

    Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse. PMID:22606329

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

    PubMed

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regulator of ABA signalling, and stimulate its ubiquitination and degradation. Genetic analysis shows that MYB96 is epistatic to MIEL1 in the control of ABA sensitivity in seeds. While MIEL1 acts primarily via MYB96 in seed germination, MIEL1 regulates protein turnover of both MYB96 and MYB30 in vegetative tissues. We find that ABA regulates the expression of MYB30-responsive genes during pathogen infection and this regulation is partly dependent on MIEL1. These results suggest that MIEL1 may facilitate crosstalk between ABA and biotic stress signalling. PMID:27615387

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

    PubMed

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

    2014-05-01

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

  17. Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

    SciTech Connect

    Kozlov, G.; Nguyen, L; Lin, T; De Crescenzo, G; Park, M; Gehring, K

    2007-01-01

    EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85{angstrom} crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices {alpha}1 and {alpha}3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

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

    PubMed Central

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

    2016-01-01

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