Activity-based in vitro selection of T4 DNA ligase

International Nuclear Information System (INIS)

Takahashi, Fumio; Funabashi, Hisakage; Mie, Masayasu; Endo, Yaeta; Sawasaki, Tatsuya; Aizawa, Masuo; Kobatake, Eiry

2005-01-01

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA

A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.

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Rosebrock, Tracy R; Zeng, Lirong; Brady, Jennifer J; Abramovitch, Robert B; Xiao, Fangming; Martin, Gregory B

2007-07-19

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

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Leng, Feng; Yu, Jiekai; Zhang, Chunxiao; Alejo, Salvador; Hoang, Nam; Sun, Hong; Lu, Fei; Zhang, Hui

2018-04-24

Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4 DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4 DCAF5 . Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4 DCAF5 . Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.

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Shao, Genbao; Wang, Ranran; Sun, Aiqin; Wei, Jing; Peng, Ke; Dai, Qian; Yang, Wannian; Lin, Qiong

2018-02-19

EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4

Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase: DAWIDZIAK et al.

Energy Technology Data Exchange (ETDEWEB)

Dawidziak, Daria M. [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville Virginia; Sanchez, Jacint G. [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville Virginia; Wagner, Jonathan M. [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville Virginia; Ganser-Pornillos, Barbie K. [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville Virginia; Pornillos, Owen [Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville Virginia

2017-07-24

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. An emerging general model is that TRIMs form elongated antiparallel coiled-coil dimers that prevent interaction of the two attendant RING domains. The RING domains themselves bind E2 conjugating enzymes as dimers, implying that an active TRIM ligase requires higher-order oligomerization of the basal coiled-coil dimers. Here, we report crystal structures of the TRIM23 RING domain in isolation and in complex with an E2–ubiquitin conjugate. Our results indicate that TRIM23 enzymatic activity requires RING dimerization, consistent with the general model of TRIM activation.

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

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van den Boomen, Dick J H; Timms, Richard T; Grice, Guinevere L; Stagg, Helen R; Skødt, Karsten; Dougan, Gordon; Nathan, James A; Lehner, Paul J

2014-08-05

The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component of a novel mammalian ERAD complex.

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

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Liu, Feng; Wang, Xiao; Su, Mengying; Yu, Mengyuan; Zhang, Shengchun; Lai, Jianbin; Yang, Chengwei; Wang, Yaqin

2015-09-17

SUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium. In Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1. Our results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.

Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

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Sarkar, Sukumar; Brautigan, David L; Larner, James M

2017-08-01

Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR . ©2017 American Association for Cancer Research.

The APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function.

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Wan, Lixin; Chen, Ming; Cao, Juxiang; Dai, Xiangpeng; Yin, Qing; Zhang, Jinfang; Song, Su-Jung; Lu, Ying; Liu, Jing; Inuzuka, Hiroyuki; Katon, Jesse M; Berry, Kelsey; Fung, Jacqueline; Ng, Christopher; Liu, Pengda; Song, Min Sup; Xue, Lian; Bronson, Roderick T; Kirschner, Marc W; Cui, Rutao; Pandolfi, Pier Paolo; Wei, Wenyi

2017-04-01

BRAF drives tumorigenesis by coordinating the activation of the RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathways governing BRAF kinase activity and protein stability remain undefined. Here, we report that in primary cells with active APC FZR1 , APC FZR1 earmarks BRAF for ubiquitination-mediated proteolysis, whereas in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APC FZR1 , leading to elevation of a cohort of oncogenic APC FZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APC FZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion cooperates with AKT hyperactivation to transform primary melanocytes, whereas genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant coactivation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis. Significance: FZR1 inhibits BRAF oncogenic functions via both APC-dependent proteolysis and APC-independent disruption of BRAF dimers, whereas hyperactivated ERK and CDK4 reciprocally suppress APC FZR1 E3 ligase activity. Aberrancies in this newly defined signaling network might account for BRAF hyperactivation in human cancers, suggesting that targeting CYCLIN D1/CDK4, alone or in combination with BRAF/MEK inhibition, can be an effective anti-melanoma therapy. Cancer Discov; 7(4); 424-41. ©2017 AACR. See related commentary by Zhang and Bollag, p. 356 This article is highlighted in the In This Issue feature, p. 339 . ©2017 American Association for Cancer Research.

Mild pituitary phenotype in 3- and 12-month-old Aip-deficient male mice.

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Lecoq, Anne-Lise; Zizzari, Philippe; Hage, Mirella; Decourtye, Lyvianne; Adam, Clovis; Viengchareun, Say; Veldhuis, Johannes D; Geoffroy, Valérie; Lombès, Marc; Tolle, Virginie; Guillou, Anne; Karhu, Auli; Kappeler, Laurent; Chanson, Philippe; Kamenický, Peter

2016-10-01

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas, particularly of the somatotroph lineage. Mice with global heterozygous inactivation of Aip (Aip(+/-)) also develop pituitary adenomas but differ from AIP-mutated patients by the high penetrance of pituitary disease. The endocrine phenotype of these mice is unknown. The aim of this study was to determine the endocrine phenotype of Aip(+/-) mice by assessing the somatic growth, ultradian pattern of GH secretion and IGF1 concentrations of longitudinally followed male mice at 3 and 12 months of age. As the early stages of pituitary tumorigenesis are controversial, we also studied the pituitary histology and somatotroph cell proliferation in these mice. Aip(+/-) mice did not develop gigantism but exhibited a leaner phenotype than wild-type mice. Analysis of GH pulsatility by deconvolution in 12-month-old Aip(+/-) mice showed a mild increase in total GH secretion, a conserved GH pulsatility pattern, but a normal IGF1 concentration. No pituitary adenomas were detected up to 12 months of age. An increased ex vivo response to GHRH of pituitary explants from 3-month-old Aip(+/-) mice, together with areas of enlarged acini identified on reticulin staining in the pituitary of some Aip(+/-) mice, was suggestive of somatotroph hyperplasia. Global heterozygous Aip deficiency in mice is accompanied by subtle increase in GH secretion, which does not result in gigantism. The absence of pituitary adenomas in 12-month-old Aip(+/-) mice in our experimental conditions demonstrates the important phenotypic variability of this congenic mouse model. © 2016 Society for Endocrinology.

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

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Mirna Perusina Lanfranca

2014-05-01

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

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

DEFF Research Database (Denmark)

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

2008-01-01

of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis......The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...... analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s...

Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

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Lemak, Alexander; Yee, Adelinda [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health Center, Department of Molecular Microbial and Structural Biology (United States); Dhe-Paganon, Sirano, E-mail: sirano.dhepaganon@utoronto.ca [University of Toronto, Structural Genomics Consortium (Canada); Arrowsmith, Cheryl H., E-mail: carrow@uhnresearch.ca [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada)

2011-09-15

Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X{sub 4}-Cys-X{sub 4}-Cys-X{sub 28}-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two {alpha}-helicies.

Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

International Nuclear Information System (INIS)

Lemak, Alexander; Yee, Adelinda; Bezsonova, Irina; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.

2011-01-01

Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X 4 -Cys-X 4 -Cys-X 28 -Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two α-helicies.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

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Pepper, Ian J; Van Sciver, Robert E; Tang, Amy H

2017-08-07

The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future

Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

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Hirotaka Takahashi

Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

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

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Gary T ZeRuth

Full Text Available The transcription factor Gli-similar 3 (Glis3 plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

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

International Nuclear Information System (INIS)

Tan, Can; Zhang, Li-Yang; Chen, Hong; Xiao, Ling; Liu, Xian-Peng; Zhang, Jian-Xiang

2011-01-01

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

A conserved role for the ARC1 E3 ligase in Brassicaceae self-incompatibility

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Daphne eGoring

2014-05-01

Full Text Available 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 ARM 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.

Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL)

NARCIS (Netherlands)

Sorrentino, Vincenzo; Scheer, Lilith; Santos, Ana; Reits, Eric; Bleijlevens, Boris; Zelcer, Noam

2011-01-01

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR).

SAG/ROC-SCFβ-TrCP E3 Ubiquitin Ligase Promotes Pro-Caspase-3 Degradation as a Mechanism of Apoptosis Protection

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Mingjia Tan

2006-12-01

Full Text Available Skp1-cullin-F-box protein (SCF is a multicomponent E3 ubiquitin (Ub ligase that ubiquitinates a number of important biologic molecules such as p27, β-catenin, and lκB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG, as well as its family member ROC1/Rbxi, bound to the proinactive form of caspase-3 (pro-caspase-3. Binding was likely mediated through F-box protein, β-transducin repeat-containing protein (β-TrCP, which binds to the first 38 amino acids of pro-caspase-3. Importantly, β-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative β-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF -Trcp promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCFβ-TrCP E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1, or β-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCFβ-TrCP E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.

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

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

2011-12-16

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

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

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Qiang Wang

2017-03-01

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

COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function Through its E3 Ubiquitin Ligase Activity

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Joo Yong Kim

2016-08-01

Full Text Available 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.

Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

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Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

2017-12-07

The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

The MIEL1 E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis Stems.

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Lee, Hong Gil; Kim, Juyoung; Suh, Mi Chung; Seo, Pil Joon

2017-07-01

Cuticular wax is an important hydrophobic layer that covers the plant aerial surface. Cuticular wax biosynthesis is shaped by multiple layers of regulation. In particular, a pair of R2R3-type MYB transcription factors, MYB96 and MYB30, are known to be the main participants in cuticular wax accumulation. Here, we report that the MYB30-INTERACTING E3 LIGASE 1 (MIEL1) E3 ubiquitin ligase controls the protein stability of the two MYB transcription factors and thereby wax biosynthesis in Arabidopsis. MIEL1-deficient miel1 mutants exhibit increased wax accumulation in stems, with up-regulation of wax biosynthetic genes targeted by MYB96 and MYB30. Genetic analysis reveals that wax accumulation of the miel1 mutant is compromised by myb96 or myb30 mutation, but MYB96 is mainly epistatic to MIEL1, playing a predominant role in cuticular wax deposition. These observations indicate that the MIEL1-MYB96 module is important for balanced cuticular wax biosynthesis in developing inflorescence stems. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements.

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Jason Liang

2018-03-01

Full Text Available Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO. Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.

RING E3 ligases: key regulatory elements are involved in abiotic stress responses in plants.

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Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum; Hong, Jeong Soo; Oh, Tae Rin; Kim, Woo Taek; Yang, Seong Wook

2017-08-01

Plants are constantly exposed to a variety of abiotic stresses, such as drought, heat, cold, flood, and salinity. To survive under such unfavorable conditions, plants have evolutionarily developed their own resistant-mechanisms. For several decades, many studies have clarified specific stress response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants. [BMB Reports 2017; 50(8): 393-400].

Structure of a Glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface

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Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.; Hammel, Michal; Lambert, Lester J.; Waddell, M. Brett; Mittag, Tanja; DeCaprio, James A.; Schulman, Brenda A.

2012-01-01

Summary The ~300 human Cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1’s RING domain, regulates the RBX1-CUL1-containing SCFFBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN’s selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition. PMID:22748924

Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase.

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Ishimoto, Kenji; Hayase, Ayaka; Kumagai, Fumiko; Kawai, Megumi; Okuno, Hiroko; Hino, Nobumasa; Okada, Yoshiaki; Kawamura, Takeshi; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Tachibana, Keisuke; Doi, Takefumi

2017-06-17

Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes. Copyright © 2017 Elsevier Inc. All rights reserved.

SCFJFK is a bona fide E3 ligase for ING4 and a potent promoter of the angiogenesis and metastasis of breast cancer

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

2015-01-01

Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1–Cul1–F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCFJFK as a bona fide E3 ligase for ING4 and unraveled the JFK–ING4–NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention. PMID:25792601

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

International Nuclear Information System (INIS)

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

2013-01-01

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

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

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Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells.

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Paolino, Magdalena; Choidas, Axel; Wallner, Stephanie; Pranjic, Blanka; Uribesalgo, Iris; Loeser, Stefanie; Jamieson, Amanda M; Langdon, Wallace Y; Ikeda, Fumiyo; Fededa, Juan Pablo; Cronin, Shane J; Nitsch, Roberto; Schultz-Fademrecht, Carsten; Eickhoff, Jan; Menninger, Sascha; Unger, Anke; Torka, Robert; Gruber, Thomas; Hinterleitner, Reinhard; Baier, Gottfried; Wolf, Dominik; Ullrich, Axel; Klebl, Bert M; Penninger, Josef M

2014-03-27

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

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

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Da-Wei Xin

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

AIP Member Societies Entering the 1980's. AIP Report.

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Porter, Beverly Fearn

Information is presented on the composition of the 1979 membership of the American Institute of Physics (AIP). Questionnaires were administered to members of the nine independent societies of the AIP to determine demographic and employment information. Background characteristics of the membership include the following: more than half identify…

AIP mutations and gigantism.

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Rostomyan, Liliya; Potorac, Iulia; Beckers, Pablo; Daly, Adrian F; Beckers, Albert

2017-06-01

AIP mutations are rare in sporadic acromegaly but they are seen at a higher frequency among certain specific populations of pituitary adenoma patients (pituitary gigantism cases, familial isolated pituitary adenoma (FIPA) kindreds, and patients with macroadenomas who are diagnosed ≤30 years). AIP mutations are most prevalent in patients with pituitary gigantism (29% of this group were found to have mutations in AIP gene). These data support targeted genetic screening for AIP mutations/deletions in these groups of pituitary adenoma patients. Earlier diagnosis of AIP-related acromegaly-gigantism cases enables timely clinical evaluation and treatment, thereby improving outcomes in terms of excessive linear growth and acromegaly comorbidities. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

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

2015-03-15

Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1-Cul1-F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCF(JFK) as a bona fide E3 ligase for ING4 and unraveled the JFK-ING4-NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention. © 2015 Yan et al.; Published by Cold Spring Harbor Laboratory Press.

Advanced Information Processing System (AIPS)

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Pitts, Felix L.

1993-01-01

Advanced Information Processing System (AIPS) is a computer systems philosophy, a set of validated hardware building blocks, and a set of validated services as embodied in system software. The goal of AIPS is to provide the knowledgebase which will allow achievement of validated fault-tolerant distributed computer system architectures, suitable for a broad range of applications, having failure probability requirements of 10E-9 at 10 hours. A background and description is given followed by program accomplishments, the current focus, applications, technology transfer, FY92 accomplishments, and funding.

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

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

2010-01-12

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

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

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Jana Kamanova

2016-04-01

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

Structure of the Siz/PIAS SUMO E3 ligase Siz1 and determinants required for SUMO modification of PCNA

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Yunus, Ali A.; Lima, Christopher D.

2009-01-01

Summary 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 E2~SUMO thioester while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a non-consensus 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. PMID:19748360

March1 E3 Ubiquitin Ligase Modulates Features of Allergic Asthma in an Ovalbumin-Induced Mouse Model of Lung Inflammation

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Osama A. Kishta

2018-01-01

Full Text Available Membrane-associated RING-CH-1 (March1 is a member of the March family of E3 ubiquitin ligases. March1 downregulates cell surface expression of MHC II and CD86 by targeting them to lysosomal degradation. Given the key roles of MHC class II and CD86 in T cell activation and to get further insights into the development of allergic inflammation, we asked whether March1 deficiency exacerbates or attenuates features of allergic asthma in mice. Herein, we used an acute model of allergy to compare the asthmatic phenotype of March1-deficient and -sufficient mice immunized with ovalbumin (OVA and later challenged by intranasal instillation of OVA in the lungs. We found that eosinophilic inflammation in airways and lung tissue was similar between WT and March1−/− allergic mice, whereas neutrophilic inflammation was significant only in March1−/− mice. Airway hyperresponsiveness as well as levels of IFN-γ, IL-13, IL-6, and IL-10 was lower in the lungs of asthmatic March1−/− mice compared to WT, whereas lung levels of TNF-α, IL-4, and IL-5 were not significantly different. Interestingly, in the serum, levels of total and ova-specific IgE were reduced in March1-deficient mice as compared to WT mice. Taken together, our results demonstrate a role of March1 E3 ubiquitin ligase in modulating allergic responses.

Functional Characterization of the Apple RING E3 Ligase MdMIEL1 in Transgenic Arabidopsis

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Jianping AN

2017-03-01

Full Text Available E3 ubiquitin ligases are involved in various physiological processes, and they play pivotal roles in growth and development. In this study, we identified a previously unknown gene in the apple fruit (Malus × domestica and named it MdMIEL1. The MdMIEL1 gene encoded a protein that contained a zinc-finger domain at its N-terminus and a RING-finger motif at its C-terminus. To investigate MdMIEL1 functions, we generated transgenic Arabidopsis lines expressing the MdMIEL1 gene under the control of the Cauliflower mosaic virus 35S promoter. Interestingly, ectopic expression of MdMIEL1 in Arabidopsis produced multiple phenotypes, including early germination, early flowering and a lateral root number increase relative to wild-type plants. Further analysis indicated that MdMIEL1 regulated lateral root initiation by increasing auxin accumulation in the roots. In a word, these results suggest that, MdMIEL1 as a novel RING-finger ubiquitin ligase influences plant growth and development, and highlight that MdMIEL1 regulates lateral root growth.

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

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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. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

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

Directory of Open Access Journals (Sweden)

Beatriz Orosa

2017-01-01

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

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

Science.gov (United States)

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

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

Science.gov (United States)

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

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Functional characterization of Anaphase Promoting Complex/Cyclosome (APC/C) E3 ubiquitin ligases in tumorigenesis

Science.gov (United States)

Zhang, Jinfang; Wan, Lixin; Dai, Xiangpeng; Sun, Yi; Wei, Wenyi

2014-01-01

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets. PMID:24569229

Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells

International Nuclear Information System (INIS)

Durante, M.; Grossi, G.F.; Napolitano, M.; Gialanella, G.

1991-01-01

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, the authors found that PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. They show that concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on ligase enzyme dose and on ATP concentration in the hypertonic medium. They conclude that T4 DNA ligase is able to repair some potentially lethal damage produced by restriction endonucleases in eucaryotic cells. (author)

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

DEFF Research Database (Denmark)

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

2015-01-01

and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only......Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22...

News from the Library: Focus on AIP conference proceedings

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CERN Library

2011-01-01

The American Institute of Physics (AIP) publishes conference proceedings covering a broad range of subjects, such as nuclear and particle physics, materials science, astronomy and astrophysics and many more. Today, this series includes some 1,300 volumes, starting with Volume 1 (1970), and dozens of new volumes are added each year.   This represents more than 100,000 conference papers available online. Among other things, AIP publishes e.g. the proceedings of the International Cryogenic Materials Conference, the Cryogenic Engineering Conference, and the International Workshop on Neutrino Factories and Superbeams. You can access AIP conference proceedings from CERN or from outside, thanks to the Library Proxy Service. See instructions on how to access them from outside CERN: access AIP Proceedings. Please send any feedback and questions to library.desk@cern.ch.  

IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases

DEFF Research Database (Denmark)

Schmid, Fabian Marc; Schou, Kenneth Bødtker; Vilhelm, Martin Juel

2018-01-01

ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport...... protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor...

The E3 Ubiquitin Ligase MIB-1 Is Necessary To Form the Nuclear Halo in Caenorhabditis elegans Sperm.

Science.gov (United States)

Herrera, Leslie A; Starr, Daniel A

2018-05-18

Unlike the classical nuclear envelope with two membranes found in other eukaryotic cells, most nematode sperm nuclei are not encapsulated by membranes. Instead, they are surrounded by a nuclear halo of unknown composition. How the halo is formed and regulated is unknown. We used forward genetics to identify molecular lesions behind three classical fer (fertilization defective) mutations that disrupt the ultrastructure of the Caenorhabditis elegans sperm nuclear halo. We found fer-2 and fer-4 alleles to be nonsense mutations in mib-1. fer-3 was caused by a nonsense mutation in eri-3 GFP::MIB-1 was expressed in the germline during early spermatogenesis, but not in mature sperm. mib-1 encodes a conserved E3 ubiquitin ligase homologous to vertebrate Mib1 and Mib2, which function in Notch signaling. Here, we show that mib-1 is important for male sterility and is involved in the regulation or formation of the nuclear halo during nematode spermatogenesis. Copyright © 2018, G3: Genes, Genomes, Genetics.

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

KAUST Repository

Zhao, Huayan; Zhang, Huoming; Cui, Peng; Ding, Feng; Wang, Guangchao; Li, Rongjun; Jenks, Matthew A.; Lü , Shiyou; Xiong, Liming

2014-01-01

The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young

Comparative analysis of the end-joining activity of several DNA ligases.

Directory of Open Access Journals (Sweden)

Robert J Bauer

Full Text Available DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1 DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3'- and 5'- single base overhangs, and 5'-two base overhangs. This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5'-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3'-single base overhangs and 2-base overhangs effectively with little blunt or 5'- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5'-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

Science.gov (United States)

Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

2016-02-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

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

NARCIS (Netherlands)

Hong, Cynthia; Duit, Sarah; Jalonen, Pilvi; Out, Ruud; Scheer, Lilith; Sorrentino, Vincenzo; Boyadjian, Rima; Rodenburg, Kees C. W.; Foley, Edan; Korhonen, Laura; Lindholm, Dan; Nimpf, Johannes; van Berkel, Theo J. C.; Tontonoz, Peter; Zelcer, Noam

2010-01-01

We have previously identified the E3-ubiquitin ligase Inducible Degrader of the LDLR (Idol)1 as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by Liver X Receptors (LXRs) and its expression is responsive to cellular sterol status independent of the

SUMO-targeted ubiquitin ligases.

Science.gov (United States)

Sriramachandran, Annie M; Dohmen, R Jürgen

2014-01-01

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. © 2013. Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2017-02-10

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

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

Directory of Open Access Journals (Sweden)

Wei Li

2008-01-01

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

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

Science.gov (United States)

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

2017-10-03

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

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

Directory of Open Access Journals (Sweden)

Richard T Timms

Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

Human cytomegalovirus IE1 downregulates Hes1 in neural progenitor cells as a potential E3 ubiquitin ligase.

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Xi-Juan Liu

2017-07-01

Full Text Available Congenital human cytomegalovirus (HCMV infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs. As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1 is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1 is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase.

Shutdown of interferon signaling by a viral-hijacked E3 ubiquitin ligase

Directory of Open Access Journals (Sweden)

Kaitlin A. Davis

2017-11-01

Full Text Available Viruses manipulate cellular processes to create an environment favorable to replication. For most viruses, this includes subverting the expression of interferon (IFN, a signaling molecule that can stimulate production of a vast array of antiviral gene products. Rotavirus, a segmented double-stranded RNA virus that causes acute gastroenteritis in infants and young children, inhibits IFN expression through its nonstructural protein NSP1. This viral protein stifles IFN expression by inducing the degradation of host factors that are necessary for upregulating the activity of IFN genes. In the case of nearly all human and porcine rotavirus strains, NSP1 induces the ubiquitination-dependent proteasomal degradation of β-transducin repeat containing protein (β-TrCP, a host factor that plays an essential role in activating the IFN-transcription factor, NF-κB. Key to the process is the presence of a decoy sequence (degron at the C-terminus of NSP1 that causes β-TrCP to mistakenly bind NSP1 instead of its natural target, inhibitor-of-κB (IκB. In a recent report published by Davis et al [2017; mBio 8(4: e01213-17], we describe molecular requirements that govern NSP1 recognition of β-TrCP, including an essential degron phosphorylation event, and the step-wise incorporation of NSP1 into hijacked cullin-RING E3 ligases (CRLs that ubiquitinate and tag β-TrCP for degradation. Notably, although β-TrCP is chiefly recognized for its role as a master regulator of NF-κB signaling and IFN expression, β-TrCP also controls the stability of checkpoint proteins implicated in numerous other cellular pathways with antiviral activities, including autophagy and apoptosis. Thus, the impact of NSP1 on creating an intracellular environment favorable to virus replication may extend well beyond the IFN signaling pathway.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Science.gov (United States)

Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

Directory of Open Access Journals (Sweden)

Jung Hwa Lim

Full Text Available Here, we show that E2-EPF ubiquitin carrier protein (UCP elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196. A UCP mutant in which Cys118 was changed to alanine (UCPC118A did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

The Tomato U-Box Type E3 Ligase PUB13 Acts With Group III Ubiquitin E2 Enzymes to Modulate FLS2-Mediated Immune Signaling

Directory of Open Access Journals (Sweden)

Bangjun Zhou

2018-05-01

Full Text Available In Arabidopsis and rice, the ubiquitin ligase PUB13-mediated protein degradation plays a significant role in plant pattern-triggered immunity (PTI and flowering time control. The Arabidopsis PUB13 has been shown to attenuate the pattern recognition receptor FLS2-mediated immune signaling by ubiquitinating FLS2 and consequently promoting its degradation by the 26S proteasome. Nevertheless, the cognate ubiquitin-conjugating enzymes (E2 with which PUB13 acts to modulate FLS2-mediated PTI are unknown. To address this question, we investigate here the tomato (Solanum lycopersicum homolog of PUB13, SlPUB13 by utilizing the recently characterized complete set of tomato E2s. Of the 13 groups of tomato E2s, only members in group III are found to interact and act with SlPUB13. Knocking-down of the group III E2 genes enhances callose deposition and induction of the RbohB gene in the immunity-associated, early oxidative burst after flg22 treatment. The group III E2s are also found to work with SlPUB13 to ubiquitinate FLS2 in vitro and are required for PUB13-mediated degradation of FLS2 in vivo upon flg22 treatment, suggesting an essential role for group III E2s in the modulation of FLS2-mediated immune signaling by PUB13. Additionally, another immunity-associated E3, NtCMPG1 is shown to also work specifically with members of group III E2 in the in vitro ubiquitination assay, which implies the group III E2 enzymes may cooperate with many E3 ligases to regulate different aspects of PTI. Taken together, these data corroborate the notion that group III E2 enzymes play an important role in PTI and build a foundation for further functional and mechanistic characterization of tomato PUB13.

Feline hypersomatotropism and acromegaly tumorigenesis: a potential role for the AIP gene.

Science.gov (United States)

Scudder, C J; Niessen, S J; Catchpole, B; Fowkes, R C; Church, D B; Forcada, Y

2017-04-01

Acromegaly in humans is usually sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline sequence variants of the aryl-hydrocarbon-receptor interacting protein (AIP) gene. Feline acromegaly has similarities to human acromegalic families with AIP mutations. The aim of this study was to sequence the feline AIP gene, identify sequence variants and compare the AIP gene sequence between feline acromegalic and control cats, and in acromegalic siblings. The feline AIP gene was amplified through PCR using whole blood genomic DNA from 10 acromegalic and 10 control cats, and 3 sibling pairs affected by acromegaly. PCR products were sequenced and compared with the published predicted feline AIP gene. A single nonsynonymous SNP was identified in exon 1 (AIP:c.9T > G) of two acromegalic cats and none of the control cats, as well as both members of one sibling pair. The region of this SNP is considered essential for the interaction of the AIP protein with its receptor. This sequence variant has not previously been reported in humans. Two additional synonymous sequence variants were identified (AIP:c.481C > T and AIP:c.826C > T). This is the first molecular study to investigate a potential genetic cause of feline acromegaly and identified a nonsynonymous AIP single nucleotide polymorphism in 20% of the acromegalic cat population evaluated, as well as in one of the sibling pairs evaluated. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

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Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

2017-07-01

N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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

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Shuangchen Chen

2017-06-01

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

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

KAUST Repository

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; Jin, Jing Bo

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.

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

KAUST Repository

Lin, Xiao-Li

2016-04-29

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

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

OpenAIRE

David, Diana; Jagadeeshan, Sankar; Hariharan, Ramkumar; Nair, Asha Sivakumari; Pillai, Radhakrishna Madhavan

2014-01-01

Background Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. Th...

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

Science.gov (United States)

Feeney, Laura; Muñoz, Ivan M; Lachaud, Christophe; Toth, Rachel; Appleton, Paul L; Schindler, Detlev; Rouse, John

2017-06-01

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Proposal for standardised ultrasound descriptors of abnormally invasive placenta (AIP)

DEFF Research Database (Denmark)

Collins, Sally L; Ashcroft, Anna; Braun, Thorsten

2016-01-01

on subjective interpretation of imaging signs. There is no accepted consensus on the definition of the commonly used ultrasound markers for AIP. The studies included in a recently published systematic review of antenatal sonographic diagnosis of AIP were analysed for the ultrasound descriptors. Different...... were examined for wording used to describe AIP signs. These were extracted and grouped by ultrasound modality, and synonymous or identical terms identified. The group agreed on six unified descriptors for 2D greyscale signs, four for 2D colour Doppler and one for 3D power Doppler. Four papers included...

SYVN1, an ERAD E3 Ubiquitin Ligase, Is Involved in GABAAα1 Degradation Associated with Methamphetamine-Induced Conditioned Place Preference

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Dong-Liang Jiao

2017-10-01

Full Text Available Abuse of methamphetamine (METH, a powerful addictive amphetamine-type stimulants (ATS, is becoming a global public health problem. The gamma-aminobutyric acid (GABAergic system plays a critical role in METH use disorders. By using rat METH conditioned place preference (CPP model, we previously demonstrated that METH-associated rewarding memory formation was associated with the reduction of GABAAα1 expression in the dorsal straitum (Dstr, however, the underlying mechanism was unclear. In the present study, we found that METH-induced CPP formation was accompanied by a significant increase in the expression of Synovial apoptosis inhibitor 1 (SYVN1, an endoplasmic reticulum (ER-associated degradation (ERAD E3 ubiquitin ligase, in the Dstr. The siRNA knockdown of SYVN1 significantly increased GABAAα1 protein levels in both primary cultured neurons and rodent Dstr. Inhibition of proteasomal activity by MG132 and Lactacystin significantly increased GABAAα1 protein levels. We further found that SYVN1 knockdown increased GABAAα1 in the intra-ER, but not in the extra-ER. Accordingly, endoplasmic reticulum stress (ERS-associated Glucose-regulated protein 78 (GRP78 and C/EBP homologous protein (CHOP increased. Thus, this study revealed that SYVN1, as the ERAD E3 ubiquitin ligase, was associated with Dstr GABAAα1 degradation induced by METH conditioned pairing.

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

International Nuclear Information System (INIS)

Kallijaervi, Jukka; Lahtinen, Ulla; Haemaelaeinen, Riikka; Lipsanen-Nyman, Marita; Palvimo, Jorma J.; Lehesjoki, Anna-Elina

2005-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Lu, Yong-Zhi; Sheng, Yu [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Li, Lan-Fen [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Tang, De-Wei [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liu, Xiang-Yu [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, Xiaojun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

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.

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

International Nuclear Information System (INIS)

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

2007-01-01

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 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 1 21 or P3 2 21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit

H2B ubiquitination: Conserved molecular mechanism, diverse physiologic functions of the E3 ligase during meiosis.

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Wang, Liying; Cao, Chunwei; Wang, Fang; Zhao, Jianguo; Li, Wei

2017-09-03

RNF20/Bre1 mediated H2B ubiquitination (H2Bub) has various physiologic functions. Recently, we found that H2Bub participates in meiotic recombination by promoting chromatin relaxation during meiosis. We then analyzed the phylogenetic relationships among the E3 ligase for H2Bub, its E2 Rad6 and their partner WW domain-containing adaptor with a coiled-coil (WAC) or Lge1, and found that the molecular mechanism underlying H2Bub is evolutionarily conserved from yeast to mammals. However, RNF20 has diverse physiologic functions in different organisms, which might be caused by the evolutionary divergency of their domain/motif architectures. In the current extra view, we not only elucidate the evolutionarily conserved molecular mechanism underlying H2Bub, but also discuss the diverse physiologic functions of RNF20 during meiosis.

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70.

Science.gov (United States)

Boboila, Cristian; Jankovic, Mila; Yan, Catherine T; Wang, Jing H; Wesemann, Duane R; Zhang, Tingting; Fazeli, Alex; Feldman, Lauren; Nussenzweig, Andre; Nussenzweig, Michel; Alt, Frederick W

2010-02-16

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair

International Nuclear Information System (INIS)

Ouyang, Yan; Kwon, Yong Tae; An, Jee Young; Eller, Danny; Tsai, S.-C.; Diaz-Perez, Silvia; Troke, Joshua J.; Teitell, Michael A.; Marahrens, York

2006-01-01

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2 -/- male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2 -/- embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2 -/- fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2 -/- cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2 -/- cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2 -/- cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2 -/- cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair

Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair

Energy Technology Data Exchange (ETDEWEB)

Ouyang, Yan [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Kwon, Yong Tae [Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261 (United States); An, Jee Young [Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Eller, Danny [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Tsai, S.-C. [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Diaz-Perez, Silvia [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Troke, Joshua J. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Teitell, Michael A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Marahrens, York [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States)]. E-mail: ymarahrens@mednet.ucla.edu

2006-04-11

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2{sup -/-} male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2{sup -/-} embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2{sup -/-} fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2{sup -/-} cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2{sup -/-} cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2{sup -/-} cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2{sup -/-} cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

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

Directory of Open Access Journals (Sweden)

Christina eLutz-Nicoladoni

2015-03-01

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

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

Science.gov (United States)

Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

2018-02-01

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

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

Science.gov (United States)

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

2016-01-01

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

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

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Isabelle Gillot

2009-01-01

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

Characterization of bacteriophage KVP40 and T4 RNA ligase 2

International Nuclear Information System (INIS)

Yin Shenmin; Kiong Ho, C.; Miller, Eric S.; Shuman, Stewart

2004-01-01

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a subfamily of RNA strand-joining enzymes that includes the trypanosome RNA editing ligases. A homolog of T4 Rnl2 is encoded in the 244-kbp DNA genome of vibriophage KVP40. We show that the 335-amino acid KVP40 Rnl2 is a monomeric protein that catalyzes RNA end-joining through ligase-adenylate and RNA-adenylate (AppRNA) intermediates. In the absence of ATP, pre-adenylated KVP40 Rnl2 reacts with an 18-mer 5'-PO 4 single-strand RNA (pRNA) to form an 18-mer RNA circle. In the presence of ATP, Rnl2 generates predominantly AppRNA. Isolated AppRNA can be circularized by KVP40 Rnl2 in the absence of ATP. The reactivity of phage Rnl2 and the distribution of the products are affected by the length of the pRNA substrate. Whereas 18-mer and 15-mer pRNAs undergo intramolecular sealing by T4 Rnl2 to form monomer circles, a 12-mer pRNA is ligated intermolecularly to form dimers, and a 9-mer pRNA is unreactive. In the presence of ATP, the 15-mer and 12-mer pRNAs are converted to AppRNAs, but the 9-mer pRNA is not. A single 5' deoxynucleotide substitution of an 18-mer pRNA substrate has no apparent effect on the 5' adenylation or circularization reactions of T4 Rnl2. In contrast, a single deoxyribonucleoside at the 3' terminus strongly and selectively suppresses the sealing step, thereby resulting in accumulation of high levels of AppRNA in the absence of ATP. The ATP-dependent 'capping' of RNA with AMP by Rnl2 is reminiscent of the capping of eukaryotic mRNA with GMP by GTP:RNA guanylyltransferase and suggests an evolutionary connection between bacteriophage Rnl2 and eukaryotic RNA capping enzymes

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

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Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2013-11-22

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Crystallization and preliminary X-ray crystallographic studies of VibE, a vibriobactin-specific 2,3-dihydroxybenzoate-AMP ligase from Vibrio cholerae

International Nuclear Information System (INIS)

Liu, Xiuhua; Wang, Zhi; Zhu, Deyu; Wei, Tiandi; Gu, Lichuan; Xu, Sujuan

2011-01-01

This article reports the molecular cloning, protein expression and purification, crystallization and preliminary X-ray crystallographic analysis of the vibriobactin synthetase VibE from V. cholerae. Vibriobactin synthetases (VibABCDEFH) catalyze the biosynthesis of vibriobactin in the pathogenic bacterium Vibrio cholerae. VibE, a vibriobactin-specific 2,3-dihydroxybenzoate-AMP ligase, plays a critical role in the transfer of 2,3-dihydroxybenzoate to the aryl carrier protein domain of holo VibB. Here, the cloning, protein expression and purification, crystallization and preliminary X-ray crystallographic analysis of VibE from V. cholerae are reported. The VibE crystal diffracted to 2.3 Å resolution. The crystal belonged to space group P2 1 , with unit-cell parameters a = 56.471, b = 45.927, c = 77.014 Å, β = 95.895°. There is one protein molecule in the asymmetric unit, with a corresponding Matthews coefficient of 1.63 Å 3 Da −1 and solvent content of 24.41%

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

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

2013-01-01

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

Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

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David G Nickens

Full Text Available BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxyribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2>A(1>A(3>A(4>A(0>A(6>A(8>A(10 and T(2>T(3>T(4>T(6 approximately T(1>T(8>T(10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90% joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI did not support ligation when

E3 Ubiquitin Ligase c-cbl Inhibits Microglia Activation After Chronic Constriction Injury.

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Xue, Pengfei; Liu, Xiaojuan; Shen, Yiming; Ju, Yuanyuan; Lu, Xiongsong; Zhang, Jinlong; Xu, Guanhua; Sun, Yuyu; Chen, Jiajia; Gu, Haiyan; Cui, Zhiming; Bao, Guofeng

2018-06-22

E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1β and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.

ParselTongue: AIPS Talking Python

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Kettenis, M.; van Langevelde, H. J.; Reynolds, C.; Cotton, B.

2006-07-01

After more than 20 years of service, classic AIPS still is the data reduction package of choice for many radio-interferometry projects, especially for VLBI. Its age shows, most prominently in the limited scripting capabilities of its user interface: POPS. ParselTongue is an attempt to make the trusted AIPS algorithms and AIPS data structures available in a modern dynamic programming language: Python. It also provides an environment to do distributed computing to take advantage of modern computing clusters. This makes it suitable for use as a scripting interface for doing complicated data reduction on large data sets. It is also used as a coding platform for the new calibration algorithms that are being developed for the European VLBI Network as part of the ALBUS project. Here we hope to take advantage of Python's extensive support for web-based technologies to automate things like collecting calibration data.

Sculpting ion channel functional expression with engineered ubiquitin ligases

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Kanner, Scott A; Morgenstern, Travis

2017-01-01

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels. PMID:29256394

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

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Pradeep D Uchil

2008-02-01

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

Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc

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Kühnle, Simone; Mothes, Benedikt; Matentzoglu, Konstantin; Scheffner, Martin

2013-01-01

Inactivation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with development of the Angelman syndrome. Recently, it was reported that in mice, loss of E6AP expression results in increased levels of the synaptic protein Arc and a concomitant impaired synaptic function, providing an explanation for some phenotypic features of Angelman syndrome patients. Accordingly, E6AP has been shown to negatively regulate activity-regulated cytoskeleton-associated protein (Arc) and it has been suggested that E6AP targets Arc for ubiquitination and degradation. In our study, we provide evidence that Arc is not a direct substrate for E6AP and binds only weakly to E6AP, if at all. Furthermore, we show that down-regulation of E6AP expression stimulates estradiol-induced transcription of the Arc gene. Thus, we propose that Arc protein levels are controlled by E6AP at the transcriptional rather than at the posttranslational level. PMID:23671107

The evolutionarily conserved E3 ubiquitin ligase AtCHIP contributes to plant immunity

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Xin eLi

2016-03-01

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

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

OpenAIRE

Bogdanik, Laurent P.; Sleigh, James N.; Tian, Cong; Samuels, Mark E.; Bedard, Karen; Seburn, Kevin L.; Burgess, Robert W.

2013-01-01

SUMMARY Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have rece...

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

International Nuclear Information System (INIS)

Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

2008-01-01

The biotin protein ligase from S. aureus has been overexpressed in E. coli, purified, crystallized by the hanging-drop vapour-diffusion method and analysed using X-ray diffraction. Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4 2 2 1 2, with unit-cell parameters a = b = 93.665, c = 131.95

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

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

2016-06-22

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

The Type II Hsp40 Sis1 cooperates with Hsp70 and the E3 ligase Ubr1 to promote degradation of terminally misfolded cytosolic protein.

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Daniel W Summers

Full Text Available Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP. The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response.

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Malakhova, Oxana A; Zhang, Dong-Er

2008-04-04

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate anti-viral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination.

Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

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Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

2014-01-01

Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

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

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Prabha Chandrasekaran

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

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

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Ranaweera, Ruchira S.; Yang, Xiaolu

2013-01-01

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

Acute Intermittent Porphyria (AIP)

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... the Healthcare Professionals area of our site. PBS Documentary AIP Diagnosis Stories **Diagnostic Testing for the Acute ... be administered only by physicians experienced in the management of porphyrias in a hospital setting. Panhematin is ...

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

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

2013-02-01

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

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

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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.

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

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Thorsten Pfirrmann

Full Text Available 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.

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

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

2012-02-14

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

The ubiquitin ligase SCFFBXW7α promotes GATA3 degradation.

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Song, Nan; Cao, Cheng; Tang, Yiman; Bi, Liyuan; Jiang, Yong; Zhou, Yongsheng; Song, Xin; Liu, Ling; Ge, Wenshu

2018-03-01

GATA3 is a key transcription factor in cell fate determination and its dysregulation has been implicated in various types of malignancies. However, how the abundance and function of GATA3 are regulated remains unclear. Here, we report that GATA3 is physically associated with FBXW7α, and FBXW7α destabilizes GATA3 through assembly of a SKP1-CUL1-F-box E3 ligase complex. Importantly, we showed that FBXW7α promotes GATA3 ubiquitination and degradation in a GSK3 dependent manner. Furthermore, we demonstrated that FBXW7α inhibits breast cancer cells survival through destabilizing GATA3, and the expression level of FBXW7α is negatively correlated with that of GATA3 in breast cancer samples. This study indicated that FBXW7α is a critical negative regulator of GATA3 and revealed a pathway for the maintenance of GATA3 abundance in breast cancer cells. © 2017 Wiley Periodicals, Inc.

The β-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells.

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Jumpertz, Sandra; Hennes, Thomas; Asare, Yaw; Vervoorts, Jörg; Bernhagen, Jürgen; Schütz, Anke K

2014-09-01

COP9 signalosome subunit 5 (CSN5) plays a decisive role in cellular processes such as cell cycle regulation and apoptosis via promoting protein degradation, gene transcription, and nuclear export. CSN5 regulates cullin-RING-E3 ligase (CRL) activity through its deNEDDylase function. It is overexpressed in several tumor entities, but its role in colorectal cancer (CRC) is poorly understood. Wnt/β-catenin signaling is aberrant in most CRC cells, resulting in increased levels of oncogenic β-catenin and thus tumor progression. Under physiological conditions, β-catenin levels are tightly regulated by continuous proteasomal degradation. We recently showed that knockdown of CSN5 in model and CRC cells results in decreased (phospho)-β-catenin levels. Reduced β-catenin levels were associated with an attenuated proliferation rate of different CRC cell types after CSN5 knockdown. The canonical Wnt pathway involves degradation of β-catenin by a β-TrCP1-containing E3 ligase, but is mostly non-functional in CRC cells. We thus hypothesized that alternative β-catenin degradation mediated by SIAH-1 (seven in absentia homolog-1), is responsible for the effect of CSN5 on β-catenin signaling in CRC cells. We found that SIAH-1 plays an essential role in β-catenin degradation in HCT116 CRC cells and that CSN5 affects β-catenin target gene expression in these cells. Of note, CSN5 affected SIAH-1 mRNA and SIAH-1 protein levels. Moreover, β-catenin and SIAH-1 form protein complexes with CSN5 in HCT116 cells. Lastly, we demonstrate that CSN5 promotes SIAH-1 degradation in HCT116 and SW480 cells and that this is associated with its deNEDDylase activity. In conclusion, we have identified a CSN5/β-catenin/SIAH-1 interaction network that might control β-catenin degradation in CRC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Study of charge symmetry in 4He by simultaneous 4He(e,e'p)3H and 4He(e,e'n)3He measurements

International Nuclear Information System (INIS)

Spahn, M.; Kihm, T.; Knopfle, K.T.; Friedrich, J.; Voegler, N.; Schmitt, C.; Walther, V.H.; Unkelbach, M.; Hofmann, H.M.

1989-01-01

We have measured the ( 3 H+p) and ( 3 He+n) breakup of 4 He by 4 He(e,e'c) coincidence experiments at low momentum transfer (q∼0.39 fm -1 ) in the excitation energy region 22≤E x ≤36 MeV. Both channels were studied simultaneously by detecting the charged fragments c=p, 3 H, and 3 He in the same detector system. The measured 4 He(e,e'p) 3 H and 4 He(e,e'n) 3 He angular correlations as well as the deduced total 4 He(e,e'p) 3 H and 4 He(e,e'n) 3 He cross sections are reproduced by microscopic model calculations with a charge-symmetric nuclear Hamiltonian

Kardiovaskulární riziko a AIP biomarker

Czech Academy of Sciences Publication Activity Database

Dobiášová, Milada

2010-01-01

Roč. 12, Suppl.4 (2010), s. 23-27 ISSN 1212-4184 R&D Projects: GA MZd NA6590; GA AV ČR GV306/96/K220 Institutional research plan: CEZ:AV0Z50110509 Keywords : atherosclerosis * lipoprotein profile of plasma * AIP [Log (TG/HDL-C)] Subject RIV: FQ - Public Health Care, Social Medicine

Lithium Suppresses Hedgehog Signaling via Promoting ITCH E3 Ligase Activity and Gli1–SUFU Interaction in PDA Cells

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Xinshuo Wang

2017-11-01

Full Text Available Dysregulation of Hedgehog (Hh signaling pathway is one of the hallmarks of pancreatic ductal adenocarcinoma (PDA. Lithium, a clinical mood stabilizer for the treatment of mental disorders, is known to suppress tumorigenic potential of PDA cells by targeting the Hh/Gli signaling pathway. In this study, we investigated the molecular mechanism of lithium induced down-regulation of Hh/Gli1. Our data show that lithium promotes the poly-ubiquitination and proteasome-mediated degradation of Gli1 through activating E3 ligase ITCH. Additionally, lithium enhances interaction between Gli1 and SUFU via suppressing GSK3β, which phosphorylates SUFU and destabilizes the SUFU-Gli1 inhibitory complex. Our studies illustrate a novel mechanism by which lithium suppresses Hh signaling via simultaneously promoting ITCH-dependent Gli1 ubiquitination/degradation and SUFU-mediated Gli1 inhibition.

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

OpenAIRE

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 t...

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

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Bethann McLaughlin

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

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.

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Samuel, Marcus A; Mudgil, Yashwanti; Salt, Jennifer N; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R

2008-08-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

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Rhodri M L Morgan

Full Text Available Mutations of the aryl hydrocarbon receptor interacting protein (AIP have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h mutations, R304* (nonsense mutation, R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE of TOMM20.

A novel truncating AIP mutation, p.W279*, in a familial isolated pituitary adenoma (FIPA) kindred.

Science.gov (United States)

Cansu, Güven Barış; Taşkıran, Bengür; Trivellin, Giampaolo; Faucz, Fabio R; Stratakis, Constantine A

2016-07-01

Familial isolated pituitary adenomas (FIPA) constitute 2-3% of pituitary tumours. AIP is the most commonly mutated gene in FIPA. We herein report a novel germline mutation of the AIP gene in a family with FIPA. We present two patients, a father and his 12-year-old daughter, diagnosed clinically and using laboratory measures with acromegaly-gigantism. Both underwent transsphenoidal hypophyseal surgery for macroadenomas. We initially detected a novel heterozygous germline AIP mutation, c.836G>A (p.W279*), in the father's DNA. We then found the same mutation in his affected daughter. Pituitary adenomas associated with AIP mutations mostly present as FIPA (68%) at an early age (78% occur at treatment success, and genetic counseling.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

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Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

2017-09-15

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

BRCA1 Is a Histone-H2A-Specific Ubiquitin Ligase

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Reinhard Kalb

2014-08-01

Full Text Available The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3 ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.

The AIP Model of EMDR Therapy and Pathogenic Memories

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Michael Hase

2017-09-01

Full Text Available Eye Movement Desensitization and Reprocessing (EMDR therapy has been widely recognized as an efficacious treatment for post-traumatic stress disorder (PTSD. In the last years more insight has been gained regarding the efficacy of EMDR therapy in a broad field of mental disorders beyond PTSD. The cornerstone of EMDR therapy is its unique model of pathogenesis and change: the adaptive information processing (AIP model. The AIP model developed by F. Shapiro has found support and differentiation in recent studies on the importance of memories in the pathogenesis of a range of mental disorders beside PTSD. However, theoretical publications or research on the application of the AIP model are still rare. The increasing acceptance of ideas that relate the origin of many mental disorders to the formation and consolidation of implicit dysfunctional memory lead to formation of the theory of pathogenic memories. Within the theory of pathogenic memories these implicit dysfunctional memories are considered to form basis of a variety of mental disorders. The theory of pathogenic memories seems compatible to the AIP model of EMDR therapy, which offers strategies to effectively access and transmute these memories leading to amelioration or resolution of symptoms. Merging the AIP model with the theory of pathogenic memories may initiate research. In consequence, patients suffering from such memory-based disorders may be earlier diagnosed and treated more effectively.

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

Science.gov (United States)

Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

2005-04-15

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

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Thomas R Caulfield

2014-11-01

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

The ubiquitin ligase ASB4 promotes trophoblast differentiation through the degradation of ID2.

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W H Davin Townley-Tilson

Full Text Available Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4 promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2 negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

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Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

2016-02-02

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

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

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

2017-01-01

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

The Arabidopsis E3 Ubiquitin Ligase HOS1 Negatively Regulates CONSTANS Abundance in the Photoperiodic Control of Flowering[W

Science.gov (United States)

Lazaro, Ana; Valverde, Federico; Piñeiro, Manuel; Jarillo, Jose A.

2012-01-01

The Arabidopsis thaliana early in short days6 (esd6) mutant was isolated in a screen for mutations that accelerate flowering time. Among other developmental alterations, esd6 displays early flowering in both long- and short-day conditions. Fine mapping of the mutation showed that the esd6 phenotype is caused by a lesion in the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) locus, which encodes a RING finger–containing E3 ubiquitin ligase. The esd6/hos1 mutation causes decreased FLOWERING LOCUS C expression and requires CONSTANS (CO) protein for its early flowering phenotype under long days. Moreover, CO and HOS1 physically interact in vitro and in planta, and HOS1 regulates CO abundance, particularly during the daylight period. Accordingly, hos1 causes a shift in the regular long-day pattern of expression of FLOWERING LOCUS T (FT) transcript, starting to rise 4 h after dawn in the mutant. In addition, HOS1 interacts synergistically with CONSTITUTIVE PHOTOMORPHOGENIC1, another regulator of CO protein stability, in the regulation of flowering time. Taken together, these results indicate that HOS1 is involved in the control of CO abundance, ensuring that CO activation of FT occurs only when the light period reaches a certain length and preventing precocious flowering in Arabidopsis. PMID:22408073

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

KAUST Repository

Ling, Yu

2012-01-09

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

The BRCA1 Ubiquitin ligase function sets a new trend for remodelling in DNA repair.

Science.gov (United States)

Densham, Ruth M; Morris, Joanna R

2017-03-04

The protein product of the breast and ovarian cancer gene, BRCA1, is part of an obligate heterodimer with BARD1. Together these RING bearing proteins act as an E3 ubiquitin ligase. Several functions have been attributed to BRCA1 that contribute to genome integrity but which of these, if any, require this enzymatic function was unclear. Here we review recent studies clarifying the role of BRCA1 E3 ubiquitin ligase in DNA repair. Perhaps the most surprising finding is the narrow range of BRCA1 functions this activity relates to. Remarkably ligase activity promotes chromatin remodelling and 53BP1 positioning through the remodeller SMARCAD1, but the activity is dispensable for the cellular survival in response to cisplatin or replication stressing agents. Implications for therapy response and tumor susceptibility are discussed.

Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast.

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

2017-09-29

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

A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

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Chen Yun-Ru

2012-09-01

Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

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

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Chong Wang

2016-04-01

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

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

Science.gov (United States)

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

2016-04-01

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

Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

Science.gov (United States)

Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

2008-01-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

Interaction of the deubiquitinating enzyme Ubp2 and the e3 ligase Rsp5 is required for transporter/receptor sorting in the multivesicular body pathway.

Directory of Open Access Journals (Sweden)

Mandy H Y Lam

Full Text Available Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.

TRIM32 ubiquitin E3 ligase, one enzyme for several pathologies: From muscular dystrophy to tumours.

Science.gov (United States)

Lazzari, Elisa; Meroni, Germana

2016-10-01

TRIM32 is a member of the TRIpartite Motif family characterised by the presence of an N-terminal three-domain-module that includes a RING domain, which confers E3 ubiquitin ligase activity, one or two B-box domains and a Coiled-Coil region that mediates oligomerisation. Several TRIM32 substrates were identified including muscular proteins and proteins involved in cell cycle regulation and cell motility. As ubiquitination is a versatile post-translational modification that can affect target turnover, sub-cellular localisation or activity, it is likely that diverse substrates may be differentially affected by TRIM32-mediated ubiquitination, reflecting its multi-faceted roles in muscle physiology, cancer and immunity. With particular relevance for muscle physiology, mutations in TRIM32 are associated with autosomal recessive Limb-Girdle Muscular Dystrophy 2H, a muscle-wasting disease with variable clinical spectrum ranging from almost asymptomatic to wheelchair-bound patients. In this review, we will focus on the ability of TRIM32 to mark specific substrates for proteasomal degradation discussing how the TRIM32-proteasome axis may (i) be important for muscle homeostasis and for the pathogenesis of muscular dystrophy; and (ii) define either an oncogenic or tumour suppressive role for TRIM32 in the context of different types of cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis.

Science.gov (United States)

Shah, Meera; Stebbins, John L; Dewing, Antimone; Qi, Jianfei; Pellecchia, Maurizio; Ronai, Ze'ev A

2009-12-01

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-kappaB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1alpha and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.

GEOSS AIP-2 Climate Change and Biodiversity Use Scenarios: Interoperability Infrastructures

Science.gov (United States)

Nativi, Stefano; Santoro, Mattia

2010-05-01

In the last years, scientific community is producing great efforts in order to study the effects of climate change on life on Earth. In this general framework, a key role is played by the impact of climate change on biodiversity. To assess this, several use scenarios require the modeling of climatological change impact on the regional distribution of biodiversity species. Designing and developing interoperability infrastructures which enable scientists to search, discover, access and use multi-disciplinary resources (i.e. datasets, services, models, etc.) is currently one of the main research fields for the Earth and Space Science Informatics. This presentation introduces and discusses an interoperability infrastructure which implements the discovery, access, and chaining of loosely-coupled resources in the climatology and biodiversity domains. This allows to set up and run forecast and processing models. The presented framework was successfully developed and experimented in the context of GEOSS AIP-2 (Global Earth Observation System of Systems, Architecture Implementation Pilot- Phase 2) Climate Change & Biodiversity thematic Working Group. This interoperability infrastructure is comprised of the following main components and services: a)GEO Portal: through this component end user is able to search, find and access the needed services for the scenario execution; b)Graphical User Interface (GUI): this component provides user interaction functionalities. It controls the workflow manager to perform the required operations for the scenario implementation; c)Use Scenario controller: this component acts as a workflow controller implementing the scenario business process -i.e. a typical climate change & biodiversity projection scenario; d)Service Broker implementing Mediation Services: this component realizes a distributed catalogue which federates several discovery and access components (exposing them through a unique CSW standard interface). Federated components

The E3 Ubiquitin Ligase TRIM40 Attenuates Antiviral Immune Responses by Targeting MDA5 and RIG-I

Directory of Open Access Journals (Sweden)

Chunyuan Zhao

2017-11-01

Full Text Available Retinoic acid-inducible gene-I (RIG-I-like receptors (RLRs, including melanoma differentiation-associated gene 5 (MDA5 and RIG-I, are crucial for host recognition of non-self RNAs, especially viral RNA. Thus, the expression and activation of RLRs play fundamental roles in eliminating the invading RNA viruses and maintaining immune homeostasis. However, how RLR expression is tightly regulated remains to be further investigated. In this study, we identified a major histocompatibility complex (MHC-encoded gene, tripartite interaction motif 40 (TRIM40, as a suppressor of RLR signaling by directly targeting MDA5 and RIG-I. TRIM40 binds to MDA5 and RIG-I and promotes their K27- and K48-linked polyubiquitination via its E3 ligase activity, leading to their proteasomal degradation. TRIM40 deficiency enhances RLR-triggered signaling. Consequently, TRIM40 deficiency greatly enhances antiviral immune responses and decreases viral replication in vivo. Thus, we demonstrate that TRIM40 limits RLR-triggered innate activation, suggesting TRIM40 as a potential therapeutic target for the control of viral infection.

AIP salary report

Science.gov (United States)

“1987 Salaries Society Membership Survey,” the fifth in a series of salary reports produced by the Education and Employment Statistics Division of the American Institute of Physics has recently been released. The data are based on a random sample survey of one-sixth of the U.S. and Canadian membership of the AIP Membership Societies, which include AGU, American Physical Society, Optical Society of America, Acoustical Society of America, Society of Rheology, American Association of Physics Teachers, American Crystallographic Association, American Astronomical Society, American Association of Physicists in Medicine, and the American Vacuum Society.

Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

Science.gov (United States)

Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

2015-01-01

Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction & stability and is associated with gigantism.

OpenAIRE

Salvatori, R.; Radian, S.; Diekmann, Y.; Iacovazzo, D.; David, A.; Grabovska, P.; Grassi, G.; Bussell, A-M; Stals, K.; Weber, A.; Quinton, R.; Crowne, E.; Corazzini, V.; Metherell, L. A.; Kearney, T.

2017-01-01

OBJECTIVE: Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. DESIGN & METHODS: Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to mo...

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

Science.gov (United States)

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

Science.gov (United States)

Shah, Meera; Stebbins, John L.; Dewing, Antimone; Qi, Jianfei; Pellecchia, Maurizio; Ronai, Ze’ev A.

2010-01-01

Summary The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-κB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis. PMID:19712206

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

Directory of Open Access Journals (Sweden)

Bill B. Chen

2014-04-01

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

[Mechanism of reaction catalyzed by RNA-ligase from bacteriophage T4].

Science.gov (United States)

Zagrebel'nyĭ, S N; Zernov, Iu P

1987-01-01

The dissociation constants of the complexes of RNA-ligase with acceptors, donors and the adenylylated donor A(5')ppAp have been determined on the basis of the inhibition of ATP-pyrophosphate exchange reaction. The dissociation constants of the complexes of the enzyme with "poor" acceptors (oligouridilates) have been shown to be slightly different from those with "good" acceptors (oligoadenylates). The dependence of the reaction velocity of the formation of ligation products on the concentration of acceptors (pA)4, (pU)4 and the adenylylated donor A(5)ppAp has been studied. On the basis of the data obtained the conclusion about the random addition mechanism has been drawn. The reaction takes place in the steady-state conditions in the case of (pA)4 and in the equilibrium conditions--in the case of (pU)4.

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

Science.gov (United States)

Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

2013-01-01

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

GEOSS AIP-2 Climate Change and Biodiversity Use Scenarios: Interoperability Infrastructures (Invited)

Science.gov (United States)

Nativi, S.; Santoro, M.

2009-12-01

Currently, one of the major challenges for scientific community is the study of climate change effects on life on Earth. To achieve this, it is crucial to understand how climate change will impact on biodiversity and, in this context, several application scenarios require modeling the impact of climate change on distribution of individual species. In the context of GEOSS AIP-2 (Global Earth Observation System of Systems, Architecture Implementation Pilot- Phase 2), the Climate Change & Biodiversity thematic Working Group developed three significant user scenarios. A couple of them make use of a GEOSS-based framework to study the impact of climate change factors on regional species distribution. The presentation introduces and discusses this framework which provides an interoperability infrastructures to loosely couple standard services and components to discover and access climate and biodiversity data, and run forecast and processing models. The framework is comprised of the following main components and services: a)GEO Portal: through this component end user is able to search, find and access the needed services for the scenario execution; b)Graphical User Interface (GUI): this component provides user interaction functionalities. It controls the workflow manager to perform the required operations for the scenario implementation; c)Use Scenario controller: this component acts as a workflow controller implementing the scenario business process -i.e. a typical climate change & biodiversity projection scenario; d)Service Broker implementing Mediation Services: this component realizes a distributed catalogue which federates several discovery and access components (exposing them through a unique CSW standard interface). Federated components publish climate, environmental and biodiversity datasets; e)Ecological Niche Model Server: this component is able to run one or more Ecological Niche Models (ENM) on selected biodiversity and climate datasets; f)Data Access

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli.

Science.gov (United States)

Delli-Bovi, Teegan A; Spalding, Maroya D; Prigge, Sean T

2010-10-11

Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism

OpenAIRE

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara

2017-01-01

Objective Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Design and methods Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP-region (8.3?Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to mo...

SU-E-T-437: Four-Dimensional Treatment Planning for Lung VMAT-SBRT

International Nuclear Information System (INIS)

Hashimoto, M; Takashina, M; Koizumi, M; Oohira, S; Ueda, Y; Miyazaki, M; Isono, M; Masaoka, A; Teshima, T

2015-01-01

Purpose: To assess optimal treatment planning approach of Volumetric Modulated Arc Therapy for lung Stereotactic Body Radiation Therapy (VMAT-SBRT). Methods: Subjects were 10 patients with lung cancer who had undergone 4DCT. The internal target volume (ITV) volume ranged from 2.6 to 16.5cm 3 and the tumor motion ranged from 0 to 2cm. From 4DCT, which was binned into 10 respiratory phases, 4 image data sets were created; maximum intensity projection (MIP), average intensity projection (AIP), AIP with the ITV replaced by 0HU (RITV-AIP) and RITV-AIP with the planning target volume (PTV) minus the internal target volume was set to −200 HU (HR-AIP). VMAT-SBRT plans were generated on each image set for a patient. 48Gy was prescribed to 95% of PTV. The plans were recalculated on all phase images of 4DCT and the dose distributions were accumulated using a deformable image registration software MIM Maestro™ as the 4D calculated dose to the gross tumor volume (GTV). The planned dose to the ITV and 4D calculated dose to the GTV were compared. Results: In AIP plan, 10 patients average of all dose parameters (D1%, D-mean, and D99%) discrepancy were 1Gy or smaller. MIP and RITV-AIP plans resulted in having common tendency and larger discrepancy than AIP plan. The 4D dose was lower than the planned dose, and 10 patients average of all dose parameters discrepancy were in range 1.3 to 2.6Gy. HR-AIP plan had the largest discrepancy in our trials. 4D calculated D1%, D-mean, and D99% were resulted in 3.0, 4.1, and 6.1Gy lower than the expected in plan, respectively. Conclusion: For all patients, the dose parameters expected in AIP plan approximated to 4D calculated. Using AIP image set seems optimal treatment planning approach of VMAT-SBRT for a mobile tumor. Funding Support: This work was supported by the Japan Society for the Promotion of Science Core-to-Core program (No. 23003)

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage.

Science.gov (United States)

Hu, Jian; McCall, Chad M; Ohta, Tomohiko; Xiong, Yue

2004-10-01

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

Science.gov (United States)

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

Indian Academy of Sciences (India)

Unknown

et al 2004) to study the different aspects of development and differentiation. .... In both mouse and human, two closely related proteins represent Cul4 (Cul4A and ..... from cDNA libraries representing the individual tissue con- stituents of the ...

E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression.

Science.gov (United States)

Müncheberg, Sarah; Hay, Ron T; Ip, Wing H; Meyer, Tina; Weiß, Christina; Brenke, Jara; Masser, Sawinee; Hadian, Kamyar; Dobner, Thomas; Schreiner, Sabrina

2018-04-25

HAdV E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in non-permissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 Ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established.RNF4, a cellular SUMO-targeted Ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM, and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNAi resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor, which was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 Ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain productive viral life

The E3 ubiquitin ligase RNF146 promotes colorectal cancer by activating the Wnt/β-catenin pathway via ubiquitination of Axin1.

Science.gov (United States)

Shen, Jiangli; Yu, Zhaohui; Li, Na

2018-06-20

The E3 ubiquitin ligase ring finger protein 146 (RNF146) has been implicated in tumor development. However, the role and clinical significance of RNF146 in colorectal cancer (CRC) remain unknown. In this study, we reported for the first time that RNF146 was upregulated in CRC tissues as well as in cell lines. Further, RNF146 expression was independent prognostic factor for poor outcome of CRC patients. RNF146 knockdown in cell lines inhibited cell growth, promoted cell apoptosis in vitro and suppressed colorectal tumor growth in vivo. Mechanistic investigations revealed that RNF146 exerted oncogenic role through ubiquitination of Axin1 to activate β-catenin signalling. In addition, RNF146 expression was positively correlated with β-catenin expression in CRC tissues. Collectively, our data suggest that RNF146 might function as a oncogene in human CRC, and represent a promising prognostic factor and a valuable therapeutic target for CRC. Copyright © 2018. Published by Elsevier Inc.

E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

Science.gov (United States)

Saha, Siddhartha S; Caviness, Gary; Yi, Guanghui; Raymond, Ernest L; Mbow, M Lamine; Kao, C Cheng

2018-01-01

Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R. © 2017 S. Karger AG, Basel.

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

KAUST Repository

Zhao, Huayan

2014-05-08

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

The Putative E3 Ubiquitin Ligase ECERIFERUM9 Regulates Abscisic Acid Biosynthesis and Response during Seed Germination and Postgermination Growth in Arabidopsis.

Science.gov (United States)

Zhao, Huayan; Zhang, Huoming; Cui, Peng; Ding, Feng; Wang, Guangchao; Li, Rongjun; Jenks, Matthew A; Lü, Shiyou; Xiong, Liming

2014-07-01

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

Escape from Telomere-Driven Crisis Is DNA Ligase III Dependent

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Rhiannon E. Jones

2014-08-01

Full Text Available Short dysfunctional telomeres are capable of fusion, generating dicentric chromosomes and initiating breakage-fusion-bridge cycles. Cells that escape the ensuing cellular crisis exhibit large-scale genomic rearrangements that drive clonal evolution and malignant progression. We demonstrate that there is an absolute requirement for fully functional DNA ligase III (LIG3, but not ligase IV (LIG4, to facilitate the escape from a telomere-driven crisis. LIG3- and LIG4-dependent alternative (A and classical (C nonhomologous end-joining (NHEJ pathways were capable of mediating the fusion of short dysfunctional telomeres, both displaying characteristic patterns of microhomology and deletion. Cells that failed to escape crisis exhibited increased proportions of C-NHEJ-mediated interchromosomal fusions, whereas those that escaped displayed increased proportions of intrachromosomal fusions. We propose that the balance between inter- and intrachromosomal telomere fusions dictates the ability of human cells to escape crisis and is influenced by the relative activities of A- and C-NHEJ at short dysfunctional telomeres.

A novel germline mutation in the aryl hydrocarbon receptor-interacting protein (AIP) gene in an Italian family with gigantism.

Science.gov (United States)

Urbani, C; Russo, D; Raggi, F; Lombardi, M; Sardella, C; Scattina, I; Lupi, I; Manetti, L; Tomisti, L; Marcocci, C; Martino, E; Bogazzi, F

2014-10-01

Acromegaly usually occurs as a sporadic disease, but it may be a part of familial pituitary tumor syndromes in rare cases. Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been associated with a predisposition to familial isolated pituitary adenoma. The aim of the present study was to evaluate the AIP gene in a patient with gigantism and in her relatives. Direct sequencing of AIP gene was performed in fourteen members of the family, spanning among three generations. The index case was an 18-year-old woman with gigantism due to an invasive GH-secreting pituitary adenoma and a concomitant tall-cell variant of papillary thyroid carcinoma. A novel germline mutation in the AIP gene (c.685C>T, p.Q229X) was identified in the proband and in two members of her family, who did not present clinical features of acromegaly or other pituitary disorders. Eleven subjects had no mutation in the AIP gene. Two members of the family with clinical features of acromegaly refused either the genetic or the biochemical evaluation. The Q229X mutation was predicted to generate a truncated AIP protein, lacking the last two tetratricopeptide repeat domains and the final C-terminal α-7 helix. We identified a new AIP germline mutation predicted to produce a truncated AIP protein, lacking its biological properties due to the disruption of the C-terminus binding sites for both the chaperones and the client proteins of AIP.

Sorghum Brown midrib 2 (Bmr2) gene encodes the major 4-coumarate Coenzyme A ligase involved in lignin synthesis

Science.gov (United States)

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...

New CERN DG - AIP awards and prizes

CERN Multimedia

2008-01-01

The CERN Council has appointed Rolf-Dieter Heuer to succedd Robert Aymar as CERN's director general. Yu Lu, a theoretical physics and research fellow in the institute of physics at the Chinese Academy of Sciences, is the recipient of the 2008 Tate Medal for International Leadership in Physics from the American Institute of Physics (AIP)

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

DEFF Research Database (Denmark)

Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

2010-01-01

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...... the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed...

Acute interstitial pneumonia (AIP): relationship to Hamman-Rich syndrome, diffuse alveolar damage (DAD), and acute respiratory distress syndrome (ARDS).

Science.gov (United States)

Mukhopadhyay, Sanjay; Parambil, Joseph G

2012-10-01

Acute interstitial pneumonia (AIP) is a term used for an idiopathic form of acute lung injury characterized clinically by acute respiratory failure with bilateral lung infiltrates and histologically by diffuse alveolar damage (DAD), a combination of findings previously known as the Hamman-Rich syndrome. This review aims to clarify the diagnostic criteria of AIP, its relationship with DAD and acute respiratory distress syndrome (ARDS), key etiologies that need to be excluded before making the diagnosis, and the salient clinical features. Cases that meet clinical and pathologic criteria for AIP overlap substantially with those that fulfill clinical criteria for ARDS. The main differences between AIP and ARDS are that AIP requires a histologic diagnosis of DAD and exclusion of known etiologies. AIP should also be distinguished from "acute exacerbation of IPF," a condition in which acute lung injury (usually DAD) supervenes on underlying usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF). Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

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

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Joshua R. Kane

2015-05-01

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

NMR characterization of foldedness for the production of E3 RING domains

NARCIS (Netherlands)

Huang, A.; de Jong, R.N.; Folkers, G.E.; Boelens, R.

2010-01-01

We summarize the use of NMR spectroscopy in the production and the screening of stability and foldedness of protein domains, and apply it to the RING domains of E3 ubiquitin-ligases. RING domains are involved in specific interactions with E2 ubiquitin-conjugating enzymes and thus play an essential

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

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Roland Le Borgne

2005-04-01

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

Last stop on the road to repair: structure of E. coli DNA ligase bound to nicked DNA-adenylate.

Science.gov (United States)

Nandakumar, Jayakrishnan; Nair, Pravin A; Shuman, Stewart

2007-04-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria and essential for growth. Their distinctive substrate specificity and domain organization vis-a-vis human ATP-dependent ligases make them outstanding targets for anti-infective drug discovery. We report here the 2.3 A crystal structure of Escherichia coli LigA bound to an adenylylated nick, which captures LigA in a state poised for strand closure and reveals the basis for nick recognition. LigA envelopes the DNA within a protein clamp. Large protein domain movements and remodeling of the active site orchestrate progression through the three chemical steps of the ligation reaction. The structure inspires a strategy for inhibitor design.

Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

Science.gov (United States)

Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

2008-06-01

Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

International Nuclear Information System (INIS)

Espinosa, Alexander; Oke, Vilija; Elfving, Ase; Nyberg, Filippa; Covacu, Ruxandra; Wahren-Herlenius, Marie

2008-01-01

Patients with the systemic autoimmune diseases Sjoegrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus

AIP mutations in young patients with acromegaly and the Tampico Giant: the Mexican experience.

Science.gov (United States)

Ramírez-Rentería, Claudia; Hernández-Ramírez, Laura C; Portocarrero-Ortiz, Lesly; Vargas, Guadalupe; Melgar, Virgilio; Espinosa, Etual; Espinosa-de-Los-Monteros, Ana Laura; Sosa, Ernesto; González, Baldomero; Zúñiga, Sergio; Unterländer, Martina; Burger, Joachim; Stals, Karen; Bussell, Anne-Marie; Ellard, Sian; Dang, Mary; Iacovazzo, Donato; Kapur, Sonal; Gabrovska, Plamena; Radian, Serban; Roncaroli, Federico; Korbonits, Márta; Mercado, Moisés

2016-08-01

Although aryl hydrocarbon receptor-interacting protein (AIP) mutations are rare in sporadic acromegaly, their prevalence among young patients is nonnegligible. The objectives of this study were to evaluate the frequency of AIP mutations in a cohort of Mexican patients with acromegaly with disease onset before the age of 30 and to search for molecular abnormalities in the AIP gene in teeth obtained from the "Tampico Giant". Peripheral blood DNA from 71 patients with acromegaly (51 females) with disease onset T (p.Arg304Ter), well-known truncating mutation was identified; in one of these two cases and her identical twin sister, the mutation proved to be a de novo event, since neither of their parents were found to be carriers. In the remaining three patients, new mutations were identified: a frameshift mutation (c.976_977insC, p.Gly326AfsTer), an in-frame deletion (c.872_877del, p.Val291_Leu292del) and a nonsense mutation (c.868A > T, p.Lys290Ter), which are predicted to be pathogenic based on in silico analysis. Patients with AIP mutations tended to have an earlier onset of acromegaly and harboured larger and more invasive tumours. A previously described genetic variant of unknown significance (c.869C > T, p.Ala299Val) was identified in DNA from the Tampico Giant. The prevalence of AIP mutations in young Mexican patients with acromegaly is similar to that of European cohorts. Our results support the need for genetic evaluation of patients with early onset acromegaly.

AIP mutations in Brazilian patients with sporadic pituitary adenomas: a single-center evaluation

Science.gov (United States)

Kasuki, Leandro; de Azeredo Lima, Carlos Henrique; Ogino, Liana; Camacho, Aline H S; Chimelli, Leila; Korbonits, Márta

2017-01-01

Aryl hydrocarbon receptor-interacting protein (AIP) gene mutations (AIPmut) are the most frequent germline mutations found in apparently sporadic pituitary adenomas (SPA). Our aim was to evaluate the frequency of AIPmut among young Brazilian patients with SPA. We performed an observational cohort study between 2013 and 2016 in a single referral center. AIPmut screening was carried out in 132 SPA patients with macroadenomas diagnosed up to 40 years or in adenomas of any size diagnosed until 18 years of age. Twelve tumor samples were also analyzed. Leukocyte DNA and tumor tissue DNA were sequenced for the entire AIP-coding region for evaluation of mutations. Eleven (8.3%) of the 132 patients had AIPmut, comprising 9/74 (12%) somatotropinomas, 1/38 (2.6%) prolactinoma, 1/10 (10%) corticotropinoma and no non-functioning adenomas. In pediatric patients (≤18 years), AIPmut frequency was 13.3% (2/15). Out of the 5 patients with gigantism, two had AIPmut, both truncating mutations. The Y268* mutation was described in Brazilian patients and the K273Rfs*30 mutation is a novel mutation in our patient. No somatic AIP mutations were found in the 12 tumor samples. A tumor sample from an acromegaly patient harboring the A299V AIPmut showed loss of heterozygosity. In conclusion, AIPmut frequency in SPA Brazilian patients is similar to other populations. Our study identified two mutations exclusively found in Brazilians and also shows, for the first time, loss of heterozygosity in tumor DNA from an acromegaly patient harboring the A299V AIPmut. Our findings corroborate previous observations that AIPmut screening should be performed in young patients with SPA. PMID:29074612

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

DEFF Research Database (Denmark)

Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke

2016-01-01

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...

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism.

Science.gov (United States)

Imran, Syed Ali; Aldahmani, Khaled A; Penney, Lynette; Croul, Sidney E; Clarke, David B; Collier, David M; Iacovazzo, Donato; Korbonits, Márta

2018-01-01

Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein ( AIP ) mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy. Typical presentation for a patient with AIP mutation with excess growth and eunuchoid proportions.Unusual, previously not described AIP variant with loss of the stop codon.Phenocopy may occur in families with a disease-causing germline mutation.

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

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Frederic Derbre

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

Lipid Profile and Atherogenic Index of Plasma (AIP in Vegetarians

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AN Wahida Sultana

2015-01-01

Full Text Available Background: Diet deficient in fresh fruits and vegetables are associated with an increased risk of coronary diseases. Low levels of vitamin C, vitamin E and other antioxidants may enhance the production of oxidized LDL and are important independent risk factors for coronary disease. Objective: To make a comparative evaluation of lipid profile and atherogenic index of plasma (AIP between vegetarians and nonvegetarians. Materials and Methods: This case-control study was carried out in the Department of Biochemistry of Bangabandhu Sheikh Mujib Medical University (BSMMU, Dhaka between July 2011 to June 2012. Vegetarian and nonvegetarian subjects of male sex were the study population. Vegetarians were considered as cases while nonvegetarians as controls. After proper ethical consideration a total of 30 vegetarians and 40 nonvegetarians were consecutively included in the study based on predefined inclusion and exclusion criteria. Laboratory investigations were done in the Department of Biochemistry, BSMMU, Dhaka. Results: The vegetarians had significantly lower total cholesterol and LDLcholesterol than the nonvegetarians (p=0.000 and p=0.000 respectively. Serum HDL cholesterol was also lower among the vegetarians (p=0.002 and triglycerides were almost identical in both the groups (p=0.272. Conclusion: The study reveals lower level of total cholesterol, LDLcholesterol and HDL-cholesterol in vegetarians. No difference regarding triglycerides and AIP was found between the groups. So, the findings of this study do not indicate any superiority of vegetarian diet in control and prevention of cardiac diseases.

The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

Energy Technology Data Exchange (ETDEWEB)

Miao, Min [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339 (United States); Zhu, Yunye [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Qiao, Maiju [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); Tang, Xiaofeng [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Zhao, Wei [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Xiao, Fangming [Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339 (United States); Liu, Yongsheng, E-mail: liuyongsheng1122@hfut.edu.cn [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China)

2014-08-08

Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.

The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

International Nuclear Information System (INIS)

Miao, Min; Zhu, Yunye; Qiao, Maiju; Tang, Xiaofeng; Zhao, Wei; Xiao, Fangming; Liu, Yongsheng

2014-01-01

Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato

Increased Population Risk of AIP-Related Acromegaly and Gigantism in Ireland.

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Radian, Serban; Diekmann, Yoan; Gabrovska, Plamena; Holland, Brendan; Bradley, Lisa; Wallace, Helen; Stals, Karen; Bussell, Anna-Marie; McGurren, Karen; Cuesta, Martin; Ryan, Anthony W; Herincs, Maria; Hernández-Ramírez, Laura C; Holland, Aidan; Samuels, Jade; Aflorei, Elena Daniela; Barry, Sayka; Dénes, Judit; Pernicova, Ida; Stiles, Craig E; Trivellin, Giampaolo; McCloskey, Ronan; Ajzensztejn, Michal; Abid, Noina; Akker, Scott A; Mercado, Moises; Cohen, Mark; Thakker, Rajesh V; Baldeweg, Stephanie; Barkan, Ariel; Musat, Madalina; Levy, Miles; Orme, Stephen M; Unterländer, Martina; Burger, Joachim; Kumar, Ajith V; Ellard, Sian; McPartlin, Joseph; McManus, Ross; Linden, Gerard J; Atkinson, Brew; Balding, David J; Agha, Amar; Thompson, Chris J; Hunter, Steven J; Thomas, Mark G; Morrison, Patrick J; Korbonits, Márta

2017-01-01

The aryl hydrocarbon receptor interacting protein (AIP) founder mutation R304 * (or p.R304 * ; NM_003977.3:c.910C>T, p.Arg304Ter) identified in Northern Ireland (NI) predisposes to acromegaly/gigantism; its population health impact remains unexplored. We measured R304 * carrier frequency in 936 Mid Ulster, 1,000 Greater Belfast (both in NI) and 2,094 Republic of Ireland (ROI) volunteers and in 116 NI or ROI acromegaly/gigantism patients. Carrier frequencies were 0.0064 in Mid Ulster (95%CI = 0.0027-0.013; P = 0.0005 vs. ROI), 0.001 in Greater Belfast (0.00011-0.0047) and zero in ROI (0-0.0014). R304 * prevalence was elevated in acromegaly/gigantism patients in NI (11/87, 12.6%, P acromegaly/gigantism cases. tMRCA is consistent with historical/folklore accounts of Irish giants. Forward simulations predict many undetected carriers; geographically targeted population screening improves asymptomatic carrier identification, complementing clinical testing of patients/relatives. We generated disease awareness locally, necessary for early diagnosis and improved outcomes of AIP-related disease. © 2016 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiquitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome.

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Kang, Seol-Hee; Lee, Hae-Ahm; Kim, Mina; Lee, Eunjo; Sohn, Uy Dong; Kim, Inkyeom

2017-06-01

Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1), and atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic minipumps containing ACTH (40 ng·kg -1 ·day -1 ). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, atrogin-1, and FOXO3a were upregulated and the FOXO3a promoter was targeted by the glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a were significantly decreased by the GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on the promoters. Knockdown of FOXO3a prevented dexamethasone-induced MuRF-1 and atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and atrogin-1 in Cushing's syndrome. Copyright © 2017 the American Physiological Society.

Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology.

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Malyarchuk, Svitlana; Wright, Douglas; Castore, Reneau; Klepper, Emily; Weiss, Bernard; Doherty, Aidan J; Harrison, Lynn

2007-10-01

Unlike Escherichia coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of microhomology (1-4bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: design, synthesis, crystal structures, and biological evaluation.

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Zidar, Nace; Tomašić, Tihomir; Šink, Roman; Kovač, Andreja; Patin, Delphine; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Premru, Manica Müller; Zega, Anamarija; Gobec, Stanislav; Mašič, Lucija Peterlin; Kikelj, Danijel

2011-11-01

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

The glomuvenous malformation protein Glomulin binds Rbx1 and regulates cullin RING ligase-mediated turnover of Fbw7.

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Tron, Adriana E; Arai, Takehiro; Duda, David M; Kuwabara, Hiroshi; Olszewski, Jennifer L; Fujiwara, Yuko; Bahamon, Brittany N; Signoretti, Sabina; Schulman, Brenda A; DeCaprio, James A

2012-04-13

Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM. Copyright © 2012 Elsevier Inc. All rights reserved.

AIP mutations in Brazilian patients with sporadic pituitary adenomas: a single-center evaluation

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Paula Bruna Araujo

2017-11-01

Full Text Available Aryl hydrocarbon receptor-interacting protein (AIP gene mutations (AIPmut are the most frequent germline mutations found in apparently sporadic pituitary adenomas (SPA. Our aim was to evaluate the frequency of AIPmut among young Brazilian patients with SPA. We performed an observational cohort study between 2013 and 2016 in a single referral center. AIPmut screening was carried out in 132 SPA patients with macroadenomas diagnosed up to 40 years or in adenomas of any size diagnosed until 18 years of age. Twelve tumor samples were also analyzed. Leukocyte DNA and tumor tissue DNA were sequenced for the entire AIP-coding region for evaluation of mutations. Eleven (8.3% of the 132 patients had AIPmut, comprising 9/74 (12% somatotropinomas, 1/38 (2.6% prolactinoma, 1/10 (10% corticotropinoma and no non-functioning adenomas. In pediatric patients (≤18 years, AIPmut frequency was 13.3% (2/15. Out of the 5 patients with gigantism, two had AIPmut, both truncating mutations. The Y268* mutation was described in Brazilian patients and the K273Rfs*30 mutation is a novel mutation in our patient. No somatic AIP mutations were found in the 12 tumor samples. A tumor sample from an acromegaly patient harboring the A299V AIPmut showed loss of heterozygosity. In conclusion, AIPmut frequency in SPA Brazilian patients is similar to other populations. Our study identified two mutations exclusively found in Brazilians and also shows, for the first time, loss of heterozygosity in tumor DNA from an acromegaly patient harboring the A299V AIPmut. Our findings corroborate previous observations that AIPmut screening should be performed in young patients with SPA.

Some Comments on Marketing AIP Information Products and Services.

Science.gov (United States)

King, Donald W.; Brown, A. M.

This study was addressed to marketing considerations for the American Institute of Physics (AIP) information products and services. The general system and its operation in a marketing environment, including promotion, channels of distribution and pricing are covered. Particular emphasis is placed on the cost/demand/price relationship for four…

The Putative E3 Ubiquitin Ligase ECERIFERUM9 Regulates Abscisic Acid Biosynthesis and Response during Seed Germination and Postgermination Growth in Arabidopsis1[W][OPEN

Science.gov (United States)

Zhao, Huayan; Zhang, Huoming; Cui, Peng; Ding, Feng; Wang, Guangchao; Li, Rongjun; Jenks, Matthew A.; Lü, Shiyou; Xiong, Liming

2014-01-01

The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination. PMID:24812105

Non-canonical CRL4A/4B(CDT2 interacts with RAD18 to modulate post replication repair and cell survival.

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Sarah Sertic

Full Text Available The Cullin-4(CDT2 E3 ubiquitin ligase plays an essential role in DNA replication origin licensing directing degradation of several licensing factors at the G1/S transition in order to prevent DNA re-replication. Recently a RAD18-independent role of Cullin-4(CDT2 in PCNA monoubiquitylation has been proposed. In an effort to better understand the function of Cullin-4(CDT2 E3 ubiquitin ligase in mammalian Post-Replication Repair during an unperturbed S-phase, we show that down-regulation of Cullin-4(CDT2 leads to two distinguishable independent phenotypes in human cells that unveil at least two independent roles of Cullin-4(CDT2 in S-phase. Apart from the re-replication preventing activity, we identified a non-canonical Cullin-4(CDT2 complex, containing both CUL4A and CUL4B, associated to the COP9 signalosome, that controls a RAD18-dependent damage avoidance pathway essential during an unperturbed S-phase. Indeed, we show that the non-canonical Cullin-4A/4B(CDT2 complex binds to RAD18 and it is required to modulate RAD18 protein levels onto chromatin and the consequent dynamics of PCNA monoubiquitylation during a normal S-phase. This function prevents replication stress, ATR hyper-signaling and, ultimately, apoptosis. A very similar PRR regulatory mechanism has been recently described for Spartan. Our findings uncover a finely regulated process in mammalian cells involving Post-Replication Repair factors, COP9 signalosome and a non-canonical Cullin4-based E3 ligase which is essential to tolerate spontaneous damage and for cell survival during physiological DNA replication.

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

Energy Technology Data Exchange (ETDEWEB)

Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M. (WU); (Danforth)

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.

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

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Eric R Weiss

2010-09-01

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

Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

Energy Technology Data Exchange (ETDEWEB)

Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Ding, Nan [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Qi, Yongmei; Zhang, Yingmei [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Wang, Jufang [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Huang, Dejun, E-mail: huangdj@lzu.edu.cn [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China)

2015-09-15

Highlights: • Cadmium (Cd) exposure delayed the repair of DNA damage induced by X-ray. • Cd exposure altered the phosphorylation of DNA-PKcs on Thr-2609 and Ser-2056 sites. • Cd impaired the formation of XRCC4 and Ligase IV foci, and down-regulated their protein expression. • Zinc mitigated the effects of Cd on DDR by regulating pDNA-PKcs (Thr-2609), XRCC4 and Ligase IV. - Abstract: 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 (1–8 h 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.

MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner.

Science.gov (United States)

Joshi, Shweta; Singh, Alok R; Durden, Donald L

2014-08-15

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner*

Science.gov (United States)

Joshi, Shweta; Singh, Alok R.; Durden, Donald L.

2014-01-01

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α–HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. PMID:24982421

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues

International Nuclear Information System (INIS)

Tanner, N.K.; Hanna, M.M.; Abelson, J.

1988-01-01

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs

Synthesis and structural study on (1E,2E,1'E,2'E)-3,3'-bis[(4-bromophenyl)-3,3'-(4-methy-1,2-phenylene diimine)] acetaldehyde dioxime: A combined experimental and theoretical study

Science.gov (United States)

Topal, T.; Kart, H. H.; Tunay Taşlı, P.; Karapınar, E.

2015-06-01

Tetradentate (1E,2E,1'E,2'E)-3,3'-bis[(4-bromophenyl)-3,3'-(4-methy-1,2-phenylene diimine)] acetaldehyde dioxime which possess N4 donor sets derived from the condensation of isonitroso- p-bromoacetophenone and 3,4-diaminotoluene are synthesized and characterized. The characterization of tetradentate Schiff base ligand has been deduced from LC-MS, FTIR, 13C and 1H NMR spectra and elemental analysis. Furthermore, the molecular geometry, infrared and NMR spectra of the title molecule in the ground state have been calculated by using the quantum chemical computational methods such as density functional theory (DFT) and ab initio Hartree-Fock (HF) methods with the 6-31G(d) and 6-311G(d) basis sets. The computed bond lengths and bond angles by using the both methods show the good agreement with each other. Moreover, the vibrational frequencies have been calculated and the scaled values have been compared with the experimental FTIR spectroscopic data. Assignments of the vibrational modes are made on the basis of potential energy distribution (PED) calculated from by using VEDA program. The correlations between the observed and calculated frequencies are in good agreement with each other as well as the correlation of the NMR data.

The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.

Science.gov (United States)

Han, Sangyeul; Kim, Sun; Bahl, Samira; Li, Lin; Burande, Clara F; Smith, Nicole; James, Marianne; Beauchamp, Roberta L; Bhide, Pradeep; DiAntonio, Aaron; Ramesh, Vijaya

2012-08-31

Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner.

Science.gov (United States)

David, Diana; Jagadeeshan, Sankar; Hariharan, Ramkumar; Nair, Asha Sivakumari; Pillai, Radhakrishna Madhavan

2014-01-01

Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. The purpose of the present study is to examine the effect of Smurf2 knockdown on the tumorigenic potential of human breast cancer cells emphasizing more on proliferative signaling pathway. siRNAs targeting different regions of the Smurf2 mRNA were employed to knockdown the expression of Smurf2. The biological effects of synthetic siRNAs on human breast cancer cells were investigated by examining the cell proliferation, migration, invasion, focus formation, anchorage-independent growth, cell cycle arrest, and cell cycle and cell proliferation related protein expressions upon Smurf2 silencing. Smurf2 silencing in human breast cancer cells resulted in a decreased focus formation potential and clonogenicity as well as in vitro cell migration/invasion capabilities. Moreover, knockdown of Smurf2 suppressed cell proliferation. Cell cycle analysis showed that the anti-proliferative effect of Smurf2 siRNA was mediated by arresting cells in the G0/G1 phase, which was caused by decreased expression of cyclin D1and cdk4, followed by upregulation p21 and p27. Furthermore, we demonstrated that silencing of Smurf2 downregulated the proliferation of breast cancer cells by modulating the PI3K- PTEN-AKT-FoxO3a pathway via the scaffold protein CNKSR2 which is involved in RAS-dependent signaling pathways. The present study provides the first evidence that silencing Smurf2 using synthetic siRNAs can regulate the tumorigenic properties of human breast cancer cells in a CNKSR2 dependent manner. Our results

Familial Isolated Pituitary Adenomas (FIPA) and the Pituitary Adenoma Predisposition due to Mutations in the Aryl Hydrocarbon Receptor Interacting Protein (AIP) Gene

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Aaltonen, Lauri A.; Daly, Adrian F.

2013-01-01

Pituitary adenomas are one of the most frequent intracranial tumors and occur with a prevalence of approximately 1:1000 in the developed world. Pituitary adenomas have a serious disease burden, and their management involves neurosurgery, biological therapies, and radiotherapy. Early diagnosis of pituitary tumors while they are smaller may help increase cure rates. Few genetic predictors of pituitary adenoma development exist. Recent years have seen two separate, complimentary advances in inherited pituitary tumor research. The clinical condition of familial isolated pituitary adenomas (FIPA) has been described, which encompasses the familial occurrence of isolated pituitary adenomas outside of the setting of syndromic conditions like multiple endocrine neoplasia type 1 and Carney complex. FIPA families comprise approximately 2% of pituitary adenomas and represent a clinical entity with homogeneous or heterogeneous pituitary adenoma types occurring within the same kindred. The aryl hydrocarbon receptor interacting protein (AIP) gene has been identified as causing a pituitary adenoma predisposition of variable penetrance that accounts for 20% of FIPA families. Germline AIP mutations have been shown to associate with the occurrence of large pituitary adenomas that occur at a young age, predominantly in children/adolescents and young adults. AIP mutations are usually associated with somatotropinomas, but prolactinomas, nonfunctioning pituitary adenomas, Cushing disease, and other infrequent clinical adenoma types can also occur. Gigantism is a particular feature of AIP mutations and occurs in more than one third of affected somatotropinoma patients. Study of pituitary adenoma patients with AIP mutations has demonstrated that these cases raise clinical challenges to successful treatment. Extensive research on the biology of AIP and new advances in mouse Aip knockout models demonstrate multiple pathways by which AIP may contribute to tumorigenesis. This review assesses

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

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Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

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

KAUST Repository

Park, Hyeongcheol; Kim, Hun; Koo, Sungcheol; Park, Heejin; Cheong, Misun; Hong, Hyewon; Baek, Dongwon; Chung, Woosik; Kim, Dohhoon; Bressan, Ray Anthony; Lee, Sangyeol; Bohnert, Hans Jü rgen; Yun, Daejin

2010-01-01

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

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

KAUST Repository

Park, Hyeongcheol

2010-06-18

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

An Arabidopsis E3 Ligase, SHOOT GRAVITROPISM9, Modulates the Interaction between Statoliths and F-Actin in Gravity Sensing[W][OA

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Nakamura, Moritaka; Toyota, Masatsugu; Tasaka, Masao; Morita, Miyo Terao

2011-01-01

Higher plants use the sedimentation of amyloplasts in statocytes as statolith to sense the direction of gravity during gravitropism. In Arabidopsis thaliana inflorescence stem statocyte, amyloplasts are in complex movement; some show jumping-like saltatory movement and some tend to sediment toward the gravity direction. Here, we report that a RING-type E3 ligase SHOOT GRAVITROPISM9 (SGR9) localized to amyloplasts modulates amyloplast dynamics. In the sgr9 mutant, which exhibits reduced gravitropism, amyloplasts did not sediment but exhibited increased saltatory movement. Amyloplasts sometimes formed a cluster that is abnormally entangled with actin filaments (AFs) in sgr9. By contrast, in the fiz1 mutant, an ACT8 semidominant mutant that induces fragmentation of AFs, amyloplasts, lost saltatory movement and sedimented with nearly statically. Both treatment with Latrunculin B, an inhibitor of AF polymerization, and the fiz1 mutation rescued the gravitropic defect of sgr9. In addition, fiz1 decreased saltatory movement and induced amyloplast sedimentation even in sgr9. Our results suggest that amyloplasts are in equilibrium between sedimentation and saltatory movement in wild-type endodermal cells. Furthermore, this equilibrium is the result of the interaction between amyloplasts and AFs modulated by the SGR9. SGR9 may promote detachment of amyloplasts from AFs, allowing the amyloplasts to sediment in the AFs-dependent equilibrium of amyloplast dynamics. PMID:21602290

E3 Ligase cIAP2 Mediates Downregulation of MRE11 and Radiosensitization in Response to HDAC Inhibition in Bladder Cancer.

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Nicholson, Judith; Jevons, Sarah J; Groselj, Blaz; Ellermann, Sophie; Konietzny, Rebecca; Kerr, Martin; Kessler, Benedikt M; Kiltie, Anne E

2017-06-01

The MRE11/RAD50/NBS1 (MRN) complex mediates DNA repair pathways, including double-strand breaks induced by radiotherapy. Meiotic recombination 11 homolog (MRE11) is downregulated by histone deacetylase inhibition (HDACi), resulting in reduced levels of DNA repair in bladder cancer cells and radiosensitization. In this study, we show that the mechanism of this downregulation is posttranslational and identify a C-terminally truncated MRE11, which is formed after HDAC inhibition as full-length MRE11 is downregulated. Truncated MRE11 was stabilized by proteasome inhibition, exhibited a decreased half-life after treatment with panobinostat, and therefore represents a newly identified intermediate induced and degraded in response to HDAC inhibition. The E3 ligase cellular inhibitor of apoptosis protein 2 (cIAP2) was upregulated in response to HDAC inhibition and was validated as a new MRE11 binding partner whose upregulation had similar effects to HDAC inhibition. cIAP2 overexpression resulted in downregulation and altered ubiquitination patterns of MRE11 and mediated radiosensitization in response to HDAC inhibition. These results highlight cIAP2 as a player in the DNA damage response as a posttranscriptional regulator of MRE11 and identify cIAP2 as a potential target for biomarker discovery or chemoradiation strategies in bladder cancer. Cancer Res; 77(11); 3027-39. ©2017 AACR . ©2017 American Association for Cancer Research.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

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Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-08-01

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

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Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

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

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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.

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

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Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

1997-01-01

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE from Verrucomicrobium spinosum DSM 4136(T..

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Sean E McGroty

Full Text Available Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE (E.C. 6.3.2.15 catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

An SCFFBXO28 E3 Ligase Protects Pancreatic β-Cells from Apoptosis

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Kanaka Durga Devi Gorrepati

2018-03-01

Full Text Available Loss of pancreatic β-cell function and/or mass is a central hallmark of all forms of diabetes but its molecular basis is incompletely understood. β-cell apoptosis contributes to the reduced β-cell mass in diabetes. Therefore, the identification of important signaling molecules that promote β-cell survival in diabetes could lead to a promising therapeutic intervention to block β-cell decline during development and progression of diabetes. In the present study, we identified F-box protein 28 (FBXO28, a substrate-recruiting component of the Skp1-Cul1-F-box (SCF ligase complex, as a regulator of pancreatic β-cell survival. FBXO28 was down-regulated in β-cells and in isolated human islets under diabetic conditions. Consistently, genetic silencing of FBXO28 impaired β-cell survival, and restoration of FBXO28 protected β-cells from the harmful effects of the diabetic milieu. Although FBXO28 expression positively correlated with β-cell transcription factor NEUROD1 and FBXO28 depletion also reduced insulin mRNA expression, neither FBXO28 overexpression nor depletion had any significant impact on insulin content, glucose-stimulated insulin secretion (GSIS or on other genes involved in glucose sensing and metabolism or on important β-cell transcription factors in isolated human islets. Consistently, FBXO28 overexpression did not further alter insulin content and GSIS in freshly isolated islets from patients with type 2 diabetes (T2D. Our data show that FBXO28 improves pancreatic β-cell survival under diabetogenic conditions without affecting insulin secretion, and its restoration may be a novel therapeutic tool to promote β-cell survival in diabetes.

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

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Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Comment on "Water-processed carbon nanotube/graphene hybrids with enhanced field emission properties" [AIP Advances 5, 097130 (2015)

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Rani, Reena; Bhatia, Ravi

2018-03-01

In their research paper, M. Song et al. [AIP ADVANCES 5, 097130 (2015)] have claimed to have achieved enhanced field emission (FE) characteristics of carbon nanotubes (CNT)/graphene hybrids experimentally, exhibiting improved FE parameters e.g. turn-on electric field of 0.79 V/μm, threshold electric field of 1.05 V/μm, maximum emission current density (Jmax) of 5.76 mA/cm2, and field enhancement factor (β) of ˜1.3 × 104. The authors have emphasized on the surprisingly high value of β to be the basis of their claim of achieving superior FE performance which is further attributed to the optimized mass ratio CNT/ graphene, which is 5:1 in the present case. However, the claim based upon high value of β is misleading because it does not corroborate with the obtained Jmax parameter. Also, the obtained value of J is quite low in the mentioned study as compared to the reported values. For an instance, Sameera et al. [J. Appl. Phys. 111, 044307 (2012) & Appl. Phys. Lett. 102, 033102 (2013)] have reported FE properties of CNT composites and reduced graphene oxide with Jmax and β values of the order of ˜102 mA/cm2 and 6 × 103, respectively. Therefore, the conclusions drawn by M. Song et al. [AIP ADVANCES 5, 097130 (2015)] in their paper do no hold.

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

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Laurent P. Bogdanik

2013-05-01

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

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

Science.gov (United States)

Bogdanik, Laurent P; Sleigh, James N; Tian, Cong; Samuels, Mark E; Bedard, Karen; Seburn, Kevin L; Burgess, Robert W

2013-05-01

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

Differential recruitment of DNA Ligase I and III to DNA repair sites

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Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2006-01-01

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

The biology of Mur ligases as an antibacterial target.

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Kouidmi, Imène; Levesque, Roger C; Paradis-Bleau, Catherine

2014-10-01

With antibiotic resistance mechanisms increasing in diversity and spreading among bacterial pathogens, the development of new classes of antibacterial agents against judiciously chosen targets is a high-priority task. The biochemical pathway for peptidoglycan biosynthesis is one of the best sources of antibacterial targets. Within this pathway are the Mur ligases, described in this review as highly suitable targets for the development of new classes of antibacterial agents. The amide ligases MurC, MurD, MurE and MurF function with the same catalytic mechanism and share conserved amino acid regions and structural features that can conceivably be exploited for the design of inhibitors that simultaneously target more than one enzyme. This would provide multi-target antibacterial weapons with minimized likelihood of target-mediated resistance development. © 2014 John Wiley & Sons Ltd.

Cardiac-specific ablation of the E3 ubiquitin ligase Mdm2 leads to oxidative stress, broad mitochondrial deficiency and early death.

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Ludger Hauck

Full Text Available The maintenance of normal heart function requires proper control of protein turnover. The ubiquitin-proteasome system is a principal regulator of protein degradation. Mdm2 is the main E3 ubiquitin ligase for p53 in mitotic cells thereby regulating cellular growth, DNA repair, oxidative stress and apoptosis. However, which of these Mdm2-related activities are preserved in differentiated cardiomyocytes has yet to be determined. We sought to elucidate the role of Mdm2 in the control of normal heart function. We observed markedly reduced Mdm2 mRNA levels accompanied by highly elevated p53 protein expression in the hearts of wild type mice subjected to myocardial infarction or trans-aortic banding. Accordingly, we generated conditional cardiac-specific Mdm2 gene knockout (Mdm2f/f;mcm mice. In adulthood, Mdm2f/f;mcm mice developed spontaneous cardiac hypertrophy, left ventricular dysfunction with early mortality post-tamoxifen. A decreased polyubiquitination of myocardial p53 was observed, leading to its stabilization and activation, in the absence of acute stress. In addition, transcriptomic analysis of Mdm2-deficient hearts revealed that there is an induction of E2f1 and c-Myc mRNA levels with reduced expression of the Pgc-1a/Ppara/Esrrb/g axis and Pink1. This was associated with a significant degree of cardiomyocyte apoptosis, and an inhibition of redox homeostasis and mitochondrial bioenergetics. All these processes are early, Mdm2-associated events and contribute to the development of pathological hypertrophy. Our genetic and biochemical data support a role for Mdm2 in cardiac growth control through the regulation of p53, the Pgc-1 family of transcriptional coactivators and the pivotal antioxidant Pink1.

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

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Francine C A Gérard

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

Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection

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Hodson, Charlotte; Purkiss, Andrew; Miles, Jennifer Anne; Walden, Helen

2014-01-01

Summary The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL. PMID:24389026

Land Acquisition and Relocation Assistance for Airport Improvement Program (AIP) Assisted Projects

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2017-07-10

This advisory circular (AC) provides guidance to sponsors of airport projects developed under the Airport Improvement Program (AIP) to meet the requirements of the Uniform Relocation Assistance and Real Property Acquisition Policies Act of 1970 (Pl 9...

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

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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

2012-05-01

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.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

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Penas, Clara; Ramachandran, Vimal [John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL (United States); Ayad, Nagi George, E-mail: nayad@med.miami.edu [John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL (United States); Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL (United States)

2012-01-09

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

International Nuclear Information System (INIS)

Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2012-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

Domain structure of a NHEJ DNA repair ligase from Mycobacterium tuberculosis.

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Pitcher, Robert S; Tonkin, Louise M; Green, Andrew J; Doherty, Aidan J

2005-08-19

A prokaryotic non-homologous end-joining (NHEJ) system for the repair of DNA double-strand breaks (DSBs), composed of a Ku homodimer (Mt-Ku) and a multidomain multifunctional ATP-dependent DNA ligase (Mt-Lig), has been described recently in Mycobacterium tuberculosis. Mt-Lig exhibits polymerase and nuclease activity in addition to DNA ligation activity. These functions were ascribed to putative polymerase, nuclease and ligase domains that together constitute a monomeric protein. Here, the separate polymerase, nuclease and ligase domains of Mt-Lig were cloned individually, over-expressed and the soluble proteins purified to homogeneity. The polymerase domain demonstrated DNA-dependent RNA primase activity, catalysing the synthesis of unprimed oligoribonucleotides on single-stranded DNA templates. The polymerase domain can also extend DNA in a template-dependent manner. This activity was eliminated when the catalytic aspartate residues were replaced with alanine. The ligase domain catalysed the sealing of nicked double-stranded DNA designed to mimic a DSB, consistent with the role of Mt-Lig in NHEJ. Deletion of the active-site lysine residue prevented the formation of an adenylated ligase complex and consequently thwarted ligation. The nuclease domain did not function independently as a 3'-5' exonuclease. DNA-binding assays revealed that both the polymerase and ligase domains bind DNA in vitro, the latter with considerably higher affinity. Mt-Ku directly stimulated the polymerase and nuclease activities of Mt-Lig. The polymerase domain bound Mt-Ku in vitro, suggesting it may recruit Mt-Lig to Ku-bound DNA in vivo. Consistent with these data, Mt-Ku stimulated the primer extension activity of the polymerase domain, suggestive of a functional interaction relevant to NHEJ-mediated DSB repair processes.

Biotin protein ligase from Corynebacterium glutamicum: role for growth and L: -lysine production.

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Peters-Wendisch, P; Stansen, K C; Götker, S; Wendisch, V F

2012-03-01

Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

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Tomoki Abe

2013-01-01

Full Text Available Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA, IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.

The ubiquitin ligase Cullin5^SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation

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

2017-01-01

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic pro...

3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

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Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

2017-12-01

5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.

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Das, Ushati; Chauleau, Mathieu; Ordonez, Heather; Shuman, Stewart

2014-08-05

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

Acromegaly Is More Severe in Patients With AHR or AIP Gene Variants Living in Highly Polluted Areas.

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Cannavo, S; Ragonese, M; Puglisi, S; Romeo, P D; Torre, M L; Alibrandi, A; Scaroni, C; Occhi, G; Ceccato, F; Regazzo, D; De Menis, E; Sartorato, P; Arnaldi, G; Trementino, L; Trimarchi, F; Ferrau, F

2016-04-01

An increased prevalence of acromegaly was found some years ago in a highly polluted area in North-Eastern Sicily, where high concentration of nonmethane hydrocarbons, volatile organic compounds, and cadmium was found. Aryl hydrocarbon receptor (AHR) pathway has a key role in detoxification of these compounds and in tumorigenesis. We correlated the occurrence of AHR and/or AHR-interacting protein (AIP) gene variants with acromegaly severity according to pollution exposition. This was an observational, perspective study conducted over 7 years in four Italian referral centers for pituitary diseases in which 210 patients with acromegaly were enrolled between 2008 and 2015. Genetic screening of patients for AHR and AIP variants. Clinical, biochemical, and radiological data of patients with and without AIP and/or AHR gene variants, living in polluted (high-risk for health, [HR]) or nonpolluted (NP) areas of five Italian regions were evaluated and compared. Among the 23 patients from HR areas, nine showed AHR or AIP variants. Mean IGF-I levels and pituitary tumor diameter were significantly higher in these nine patients (HR/VAR+) than in the other 14 (HR/VAR−) and in the 187 from NP areas (44 NP/VAR+). Somatostatin analogs significantly decreased mean GH and IGF-I levels in patients from NP areas and in HR/VAR− (GH P acromegaly, increased pituitary tumor size, and somatostatin analog resistance in patients living in HR areas.

A minireview of E4BP4/NFIL3 in heart failure.

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Velmurugan, Bharath Kumar; Chang, Ruey-Lin; Marthandam Asokan, Shibu; Chang, Chih-Fen; Day, Cecilia-Hsuan; Lin, Yueh-Min; Lin, Yuan-Chuan; Kuo, Wei-Wen; Huang, Chih-Yang

2018-06-01

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. The primary cause identified for HF is impaired left ventricular myocardial function, and clinical manifestations may lead to severe conditions like pulmonary congestion, splanchnic congestion, and peripheral edema. Development of new therapeutic strategies remains the need of the hour for controlling the problem of HF worldwide. Deeper insights into the molecular mechanisms involved in etiopathology of HF indicate the significant role of calcium signaling, autocrine signaling pathways, and insulin-like growth factor-1 signaling that regulates the physiologic functions of heart growth and development such as contraction, metabolism, hypertrophy, cytokine signaling, and apoptosis. In view of these facts, a transcription factor (TF) regulating the myriad of these signaling pathways may prove as a lead candidate for development of therapeutics. Adenovirus E4 promoter-binding protein (E4BP4), also known as nuclear-factor, interleukin 3 regulated (NFIL3), a type of basic leucine zipper TF, is known to regulate the signaling processes involved in the functioning of heart. The current review discusses about the expression, structure, and functional role of E4BP4 in signaling processes with emphasis on calcium signaling mechanisms, autocrine signaling, and insulin-like growth factor II receptor-mediated processes regulated by E4BP4 that may regulate the pathogenesis of HF. We propose that E4BP4, being the critical component for the regulation of the above signaling processes, may serve as a novel therapeutic target for HF, and scientific investigations are merited in this direction. © 2018 Wiley Periodicals, Inc.

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation.

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Zattas, Dimitrios; Berk, Jason M; Kreft, Stefan G; Hochstrasser, Mark

2016-06-03

Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Photoelectron spectroscopy of B4O4 (-): Dual 3c-4e π hyperbonds and rhombic 4c-4e o-bond in boron oxide clusters.

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Tian, Wen-Juan; Zhao, Li-Juan; Chen, Qiang; Ou, Ting; Xu, Hong-Guang; Zheng, Wei-Jun; Zhai, Hua-Jin; Li, Si-Dian

2015-04-07

Gas-phase anion photoelectron spectroscopy (PES) is combined with global structural searches and electronic structure calculations at the hybrid Becke 3-parameter exchange functional and Lee-Yang-Parr correlation functional (B3LYP) and single-point coupled-cluster with single, double, and perturbative triple excitations (CCSD(T)) levels to probe the structural and electronic properties and chemical bonding of the B4O4 (0/-) clusters. The measured PES spectra of B4O4 (-) exhibit a major band with the adiabatic and vertical detachment energies (ADE and VDE) of 2.64 ± 0.10 and 2.81 ± 0.10 eV, respectively, as well as a weak peak with the ADE and VDE of 1.42 ± 0.08 and 1.48 ± 0.08 eV. The former band proves to correspond to the Y-shaped global minimum of Cs B4O4 (-) ((2)A″), with the calculated ADE/VDE of 2.57/2.84 eV at the CCSD(T) level, whereas the weak band is associated with the second lowest-energy, rhombic isomer of D2h B4O4 (-) ((2)B2g) with the predicted ADE/VDE of 1.43/1.49 eV. Both anion structures are planar, featuring a B atom or a B2O2 core bonded with terminal BO and/or BO2 groups. The same Y-shaped and rhombic structures are also located for the B4O4 neutral cluster, albeit with a reversed energy order. Bonding analyses reveal dual three-center four-electron (3c-4e) π hyperbonds in the Y-shaped B4O4 (0/-) clusters and a four-center four-electron (4c-4e) π bond, that is, the so-called o-bond in the rhombic B4O4 (0/-) clusters. This work is the first experimental study on a molecular system with an o-bond.

An unusual case of an ACTH-secreting macroadenoma with a germline variant in the aryl hydrocarbon receptor-interacting protein (AIP) gene

DEFF Research Database (Denmark)

Dinesen, Pia T; Dal, Jakob; Gabrovska, Plamena

2015-01-01

growth rather than symptoms of hypersecretion. The particular AIP gene variant identified in our patient is shared by four other reported cases of CD. Future studies are needed to assess whether the reported AIP gene variant is more than just coincidental. LEARNING POINTS: CD is occasionally dominated...

Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

International Nuclear Information System (INIS)

Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi

2015-01-01

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction

Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells

Energy Technology Data Exchange (ETDEWEB)

Kanai, Dai; Ueda, Atsushi; Akagi, Tadayuki; Yokota, Takashi; Koide, Hiroshi, E-mail: hkoide@med.kanazawa-u.ac.jp

2015-04-10

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth. - Highlights: • Oct3/4 positively regulates E2F3a expression in ES cells. • Oct3/4 binds to the promoter region of the E2F3a gene. • Overexpression of E2F6, an inhibitor of E2F3a, reduces ES cell growth. • E2F3a recovers growth retardation of ES cells caused by Oct3/4 reduction.

(E-3-(4-Bromophenyl-1-(3,4-dichlorophenylprop-2-en-1-one

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Rajni Kant

2009-04-01

Full Text Available The molecule of the title compound, C15H9BrCl2O, is shown to be the E isomer, with the 3,4-dichlorobenzoyl and p-bromophenyl substituents in trans positions with respect to the chalcone olefin bond. The molecule is non-planar, the two aromatic rings forming a dihedral angle of 49.58 (1°.

Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase*

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Samai, Poulami; Shuman, Stewart

2011-01-01

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3′-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3′-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3′-OH nucleoside in the catalysis of DNA 5′-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig. PMID:21335605

Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase.

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Samai, Poulami; Shuman, Stewart

2011-04-15

Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis.

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Johnson, Jacque-Lynne F A; Lu, Chenggang; Raharjo, Eko; McNally, Karen; McNally, Francis J; Mains, Paul E

2009-06-15

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.

Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice

International Nuclear Information System (INIS)

Sun, Haiyan; Li, Ying; Feng, Shengqiu; Zou, Weihua; Guo, Kai; Fan, Chunfen; Si, Shengli

2013-01-01

Highlights: ► 4CLs play important roles in both lignin and flavonoids biosynthesis. ► PA and FA are the two main substrates of 4CL (Os4CL1/3/4/5) for lignin biosynthesis. ► Os4CL2 is suggested for flavonoid formation in defense against UV radiation. -- Abstract: 4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid

Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice

Energy Technology Data Exchange (ETDEWEB)

Sun, Haiyan [National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 (China); Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); School of Biology and Food Engineering, Changshu Institute of Technology, Changshu 215500 (China); Li, Ying; Feng, Shengqiu; Zou, Weihua [National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 (China); Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Guo, Kai [National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 (China); Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Fan, Chunfen [National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 (China); Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070 (China); College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Si, Shengli [National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070 (China); Biomass and Bioenergy Research Centre, Huazhong Agricultural University, Wuhan 430070 (China); College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); and others

2013-01-18

Highlights: ► 4CLs play important roles in both lignin and flavonoids biosynthesis. ► PA and FA are the two main substrates of 4CL (Os4CL1/3/4/5) for lignin biosynthesis. ► Os4CL2 is suggested for flavonoid formation in defense against UV radiation. -- Abstract: 4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid.

Photoelectron spectroscopy of B4O4-: Dual 3c-4e π hyperbonds and rhombic 4c-4e o-bond in boron oxide clusters

Science.gov (United States)

Tian, Wen-Juan; Zhao, Li-Juan; Chen, Qiang; Ou, Ting; Xu, Hong-Guang; Zheng, Wei-Jun; Zhai, Hua-Jin; Li, Si-Dian

2015-04-01

Gas-phase anion photoelectron spectroscopy (PES) is combined with global structural searches and electronic structure calculations at the hybrid Becke 3-parameter exchange functional and Lee-Yang-Parr correlation functional (B3LYP) and single-point coupled-cluster with single, double, and perturbative triple excitations (CCSD(T)) levels to probe the structural and electronic properties and chemical bonding of the B4O40/- clusters. The measured PES spectra of B4O4- exhibit a major band with the adiabatic and vertical detachment energies (ADE and VDE) of 2.64 ± 0.10 and 2.81 ± 0.10 eV, respectively, as well as a weak peak with the ADE and VDE of 1.42 ± 0.08 and 1.48 ± 0.08 eV. The former band proves to correspond to the Y-shaped global minimum of Cs B4O4- (2A″), with the calculated ADE/VDE of 2.57/2.84 eV at the CCSD(T) level, whereas the weak band is associated with the second lowest-energy, rhombic isomer of D2h B4O4- (2B2g) with the predicted ADE/VDE of 1.43/1.49 eV. Both anion structures are planar, featuring a B atom or a B2O2 core bonded with terminal BO and/or BO2 groups. The same Y-shaped and rhombic structures are also located for the B4O4 neutral cluster, albeit with a reversed energy order. Bonding analyses reveal dual three-center four-electron (3c-4e) π hyperbonds in the Y-shaped B4O40/- clusters and a four-center four-electron (4c-4e) π bond, that is, the so-called o-bond in the rhombic B4O40/- clusters. This work is the first experimental study on a molecular system with an o-bond.

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

Science.gov (United States)

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

A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

Directory of Open Access Journals (Sweden)

Andrew L Mammen

Full Text Available Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish, UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

Energy Technology Data Exchange (ETDEWEB)

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.

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

DEFF Research Database (Denmark)

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

2012-01-01

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation....... Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks....... Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation...

Photoelectron spectroscopy of B4O4−: Dual 3c-4e π hyperbonds and rhombic 4c-4e o-bond in boron oxide clusters

International Nuclear Information System (INIS)

Tian, Wen-Juan; Chen, Qiang; Ou, Ting; Li, Si-Dian; Zhao, Li-Juan; Xu, Hong-Guang; Zheng, Wei-Jun; Zhai, Hua-Jin

2015-01-01

Gas-phase anion photoelectron spectroscopy (PES) is combined with global structural searches and electronic structure calculations at the hybrid Becke 3-parameter exchange functional and Lee-Yang-Parr correlation functional (B3LYP) and single-point coupled-cluster with single, double, and perturbative triple excitations (CCSD(T)) levels to probe the structural and electronic properties and chemical bonding of the B 4 O 4 0/− clusters. The measured PES spectra of B 4 O 4 − exhibit a major band with the adiabatic and vertical detachment energies (ADE and VDE) of 2.64 ± 0.10 and 2.81 ± 0.10 eV, respectively, as well as a weak peak with the ADE and VDE of 1.42 ± 0.08 and 1.48 ± 0.08 eV. The former band proves to correspond to the Y-shaped global minimum of C s B 4 O 4 − ( 2 A″), with the calculated ADE/VDE of 2.57/2.84 eV at the CCSD(T) level, whereas the weak band is associated with the second lowest-energy, rhombic isomer of D 2h B 4 O 4 − ( 2 B 2g ) with the predicted ADE/VDE of 1.43/1.49 eV. Both anion structures are planar, featuring a B atom or a B 2 O 2 core bonded with terminal BO and/or BO 2 groups. The same Y-shaped and rhombic structures are also located for the B 4 O 4 neutral cluster, albeit with a reversed energy order. Bonding analyses reveal dual three-center four-electron (3c-4e) π hyperbonds in the Y-shaped B 4 O 4 0/− clusters and a four-center four-electron (4c-4e) π bond, that is, the so-called o-bond in the rhombic B 4 O 4 0/− clusters. This work is the first experimental study on a molecular system with an o-bond

Purification and biochemical characterization of Mur ligases from Staphylococcus aureus.

Science.gov (United States)

Patin, Delphine; Boniface, Audrey; Kovač, Andreja; Hervé, Mireille; Dementin, Sébastien; Barreteau, Hélène; Mengin-Lecreulx, Dominique; Blanot, Didier

2010-12-01

The Mur ligases (MurC, MurD, MurE and MurF) catalyze the stepwise synthesis of the UDP-N-acetylmuramoyl-pentapeptide precursor of peptidoglycan. The murC, murD, murE and murF genes from Staphylococcus aureus, a major pathogen, were cloned and the corresponding proteins were overproduced in Escherichia coli and purified as His(6)-tagged forms. Their biochemical properties were investigated and compared to those of the E. coli enzymes. Staphylococcal MurC accepted L-Ala, L-Ser and Gly as substrates, as the E. coli enzyme does, with a strong preference for L-Ala. S. aureus MurE was very specific for L-lysine and in particular did not accept meso-diaminopimelic acid as a substrate. This mirrors the E. coli MurE specificity, for which meso-diaminopimelic acid is the preferred substrate and L-lysine a very poor one. S. aureus MurF appeared less specific and accepted both forms (L-lysine and meso-diaminopimelic acid) of UDP-MurNAc-tripeptide, as the E. coli MurF does. The inverse and strict substrate specificities of the two MurE orthologues is thus responsible for the presence of exclusively meso-diaminopimelic acid and L-lysine at the third position of the peptide in the peptidoglycans of E. coli and S. aureus, respectively. The specific activities of the four Mur ligases were also determined in crude extracts of S. aureus and compared to cell requirements for peptidoglycan biosynthesis. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

Science.gov (United States)

Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2011-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy. PMID:22655255

Serum Immunoglobulin G4 in Discriminating Autoimmune Pancreatitis From Pancreatic Cancer: A Diagnostic Meta-analysis.

Science.gov (United States)

Dai, Cong; Cao, Qin; Jiang, Min; Sun, Ming-Jun

2018-03-01

Differentiation between autoimmune pancreatitis (AIP) and pancreatic cancer (PC) is a clinical challenge. Emerging published data on the accuracy of serum immunoglobulin G4 (IgG4) for the differential diagnosis between AIP and PC are inconsistent. The objective of our study was to perform a meta-analysis evaluating the clinical utility of serum IgG4 in the differential diagnosis between AIP and PC. We performed a systematic literature search of multiple electronic databases. The methodological quality of each study was assessed according to the Quality Assessment of Diagnostic Accuracy Studies checklist. Random-effects model was used to summarize the diagnostic odds ratio and other measures of accuracy. Eleven studies comprising 523 AIP patients and 771 PC patients were included in the meta-analysis. The summary estimates for serum IgG4 in distinguishing AIP from PC were as follows: diagnostic odds ratio, 57.30 (95% confidence interval [CI], 23.17-141.67); sensitivity, 0.72 (95% CI, 0.68-0.76); specificity, 0.93 (95% CI, 0.91-0.94). The area under the curve of serum IgG4 in distinguishing AIP from PC was 0.9200. Our meta-analysis found that serum IgG4 has high specificity and relatively low sensitivity in the differential diagnosis between AIP and PC. Therefore, serum IgG4 is useful in distinguishing AIP from PC.

The small ubiquitin-like modifier E3 ligase MdSIZ1 promotes anthocyanin accumulation by sumoylating MdMYB1 under low-temperature conditions in apple.

Science.gov (United States)

Zhou, Li-Jie; Li, Yuan-Yuan; Zhang, Rui-Fen; Zhang, Chun-Ling; Xie, Xing-Bin; Zhao, Cheng; Hao, Yu-Jin

2017-10-01

MdMYB1 acts as a crucial component of the MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis in red-skinned apples (Malus domestica), but little is known about its post-translational regulation. Here, a small ubiquitin-like modifier E3 ligase MdSIZ1 was screened out as an MdMYB1-interacting protein with a yeast two-hybridization approach. The interaction between MdSIZ1 and MdMYB1 was further verified with pull-down and CoIP assays. Furthermore, it was found that MdSIZ1 directly sumoylated MdMYB1 proteins in vivo and in vitro, especially under moderately low temperature (17 °C) conditions, and that this sumoylation was required for MdMYB1 protein stability. Moreover, the transcription level of MdSIZ1 gene was remarkably induced by low temperature and phosphorus deficiency, and MdSIZ1 overexpression exerted a large positive influence on anthocyanin accumulation and red fruit coloration, suggesting its important role in the regulation of anthocyanin biosynthesis under stress conditions. Our findings reveal an important role for a small ubiquitin-like modifier modification of MYB transcription factors in regulation of anthocyanin biosynthesis in plants. © 2017 John Wiley & Sons Ltd.

In-frame seven amino-acid duplication in AIP arose over the last 3000 years, disrupts protein interaction and stability and is associated with gigantism.

Science.gov (United States)

Salvatori, Roberto; Radian, Serban; Diekmann, Yoan; Iacovazzo, Donato; David, Alessia; Gabrovska, Plamena; Grassi, Giorgia; Bussell, Anna-Marie; Stals, Karen; Weber, Astrid; Quinton, Richard; Crowne, Elizabeth C; Corazzini, Valentina; Metherell, Lou; Kearney, Tara; Du Plessis, Daniel; Sinha, Ajay Kumar; Baborie, Atik; Lecoq, Anne-Lise; Chanson, Philippe; Ansorge, Olaf; Ellard, Sian; Trainer, Peter J; Balding, David; Thomas, Mark G; Korbonits, Márta

2017-09-01

Mutations in the aryl hydrocarbon receptor-interacting protein ( AIP ) gene are associated with pituitary adenoma, acromegaly and gigantism. Identical alleles in unrelated pedigrees could be inherited from a common ancestor or result from recurrent mutation events. Observational, inferential and experimental study, including: AIP mutation testing; reconstruction of 14 AIP -region (8.3 Mbp) haplotypes; coalescent-based approximate Bayesian estimation of the time to most recent common ancestor (tMRCA) of the derived allele; forward population simulations to estimate current number of allele carriers; proposal of mutation mechanism; protein structure predictions; co-immunoprecipitation and cycloheximide chase experiments. Nine European-origin, unrelated c.805_825dup-positive pedigrees (four familial, five sporadic from the UK, USA and France) included 16 affected (nine gigantism/four acromegaly/two non-functioning pituitary adenoma patients and one prospectively diagnosed acromegaly patient) and nine unaffected carriers. All pedigrees shared a 2.79 Mbp haploblock around AIP with additional haploblocks privately shared between subsets of the pedigrees, indicating the existence of an evolutionarily recent common ancestor, the 'English founder', with an estimated median tMRCA of 47 generations (corresponding to 1175 years) with a confidence interval (9-113 generations, equivalent to 225-2825 years). The mutation occurred in a small tandem repeat region predisposed to slipped strand mispairing. The resulting seven amino-acid duplication disrupts interaction with HSP90 and leads to a marked reduction in protein stability. The c.805_825dup allele, originating from a common ancestor, associates with a severe clinical phenotype and a high frequency of gigantism. The mutation is likely to be the result of slipped strand mispairing and affects protein-protein interactions and AIP protein stability. © 2017 The authors.

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

Science.gov (United States)

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

2014-03-07

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway*

Science.gov (United States)

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D.; Kee, Younghoon

2014-01-01

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

IgG4-Seronegative Autoimmune Pancreatitis and Sclerosing Cholangitis

Directory of Open Access Journals (Sweden)

Allon Kahn

2015-01-01

Full Text Available IgG4-related disease is a relatively novel clinical entity whose gastrointestinal manifestations include type 1 autoimmune pancreatitis (AIP and IgG4-associated sclerosing cholangitis. The presence of elevated serum IgG4 is suggestive but not essential for the diagnosis of type 1 AIP and is a pervasive feature of the proposed diagnostic criteria. The differential diagnosis of type 1 AIP includes malignant conditions, emphasizing the importance of a deliberate, comprehensive evaluation. Management of patients with a suggestive clinical presentation, but without serum IgG4 elevation, is difficult. Here we present three cases of IgG4-seronegative AIP and sclerosing cholangitis that responded to empiric steroid therapy and discuss approach considerations. These cases demonstrate the value of meticulous application of existing diagnostic algorithms to achieve a clinical diagnosis and avoid surgical intervention.

The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

Directory of Open Access Journals (Sweden)

Daniela S Silva

2013-02-01

Full Text Available AIP56 (apoptosis-inducing protein of 56 kDa is a major virulence factor of Photobacterium damselae piscicida (Phdp, a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39-Glu(40 peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

The U-box E3 ubiquitin ligase TUD1 functions with a heterotrimeric G α subunit to regulate Brassinosteroid-mediated growth in rice.

Directory of Open Access Journals (Sweden)

Xingming Hu

Full Text Available Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1 that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development.

The U-Box E3 Ubiquitin Ligase TUD1 Functions with a Heterotrimeric G α Subunit to Regulate Brassinosteroid-Mediated Growth in Rice

Science.gov (United States)

Hu, Xingming; Qian, Qian; Xu, Ting; Zhang, Yu'e; Dong, Guojun; Gao, Ting; Xie, Qi; Xue, Yongbiao

2013-01-01

Heterotrimeric G proteins are an important group of signaling molecules found in eukaryotes. They function with G-protein-coupled-receptors (GPCRs) to transduce various signals such as steroid hormones in animals. Nevertheless, their functions in plants are not well-defined. Previous studies suggested that the heterotrimeric G protein α subunit known as D1/RGA1 in rice is involved in a phytohormone gibberellin-mediated signaling pathway. Evidence also implicates D1 in the action of a second phytohormone Brassinosteroid (BR) and its pathway. However, it is unclear how D1 functions in this pathway, because so far no partner has been identified to act with D1. In this study, we report a D1 genetic interactor Taihu Dwarf1 (TUD1) that encodes a functional U-box E3 ubiquitin ligase. Genetic, phenotypic, and physiological analyses have shown that tud1 is epistatic to d1 and is less sensitive to BR treatment. Histological observations showed that the dwarf phenotype of tud1 is mainly due to decreased cell proliferation and disorganized cell files in aerial organs. Furthermore, we found that D1 directly interacts with TUD1. Taken together, these results demonstrate that D1 and TUD1 act together to mediate a BR-signaling pathway. This supports the idea that a D1-mediated BR signaling pathway occurs in rice to affect plant growth and development. PMID:23526892

ATL9, a RING zinc finger protein with E3 ubiquitin ligase activity implicated in chitin- and NADPH oxidase-mediated defense responses.

Directory of Open Access Journals (Sweden)

Marta Berrocal-Lobo

2010-12-01

Full Text Available Pathogen associated molecular patterns (PAMPs are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA, jasmonic acid (JA and ethylene (ET signaling pathways. One of these genes is ATL9 ( = ATL2G, which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET, full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst.

Spatial Representations in Local Field Potential Activity of Primate Anterior Intraparietal Cortex (AIP.

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Sebastian J Lehmann

Full Text Available The execution of reach-to-grasp movements in order to interact with our environment is an important subset of the human movement repertoire. To coordinate such goal-directed movements, information about the relative spatial position of target and effector (in this case the hand has to be continuously integrated and processed. Recently, we reported the existence of spatial representations in spiking-activity of the cortical fronto-parietal grasp network (Lehmann & Scherberger 2013, and in particular in the anterior intraparietal cortex (AIP. To further investigate the nature of these spatial representations, we explored in two rhesus monkeys (Macaca mulatta how different frequency bands of the local field potential (LFP in AIP are modulated by grip type, target position, and gaze position, during the planning and execution of reach-to-grasp movements. We systematically varied grasp type, spatial target, and gaze position and found that both spatial and grasp information were encoded in a variety of frequency bands (1-13Hz, 13-30Hz, 30-60Hz, and 60-100Hz, respectively. Whereas the representation of grasp type strongly increased towards and during movement execution, spatial information was represented throughout the task. Both spatial and grasp type representations could be readily decoded from all frequency bands. The fact that grasp type and spatial (reach information was found not only in spiking activity, but also in various LFP frequency bands of AIP, might significantly contribute to the development of LFP-based neural interfaces for the control of upper limb prostheses.

IgG4-related disease

DEFF Research Database (Denmark)

Detlefsen, Sönke; Klöppel, Günter

2018-01-01

disease (IgG4-RD). The histologic key findings are lymphoplasmacytic infiltration rich in IgG4-positive plasma cells combined with storiform fibrosis and obliterative phlebitis. Among the organs mainly affected by IgG4-RD are the pancreas and the extrahepatic bile ducts. The pancreatic and biliary...... alterations have been described under the terms autoimmune pancreatitis (AIP) and sclerosing cholangitis, respectively. These diseases are currently more precisely called IgG4-related pancreatitis (or type 1 AIP to distinguish it from type 2 AIP that is unrelated to IgG4-RD) and IgG4-related sclerosing...... cholangitis (IgG4-related SC). Clinically and grossly, both diseases commonly imitate pancreatic and biliary adenocarcinoma, tumors that are well known for their dismal prognosis. As IgG4-RD responds to steroid treatment, making a resection of a suspected tumor unnecessary, a biopsy is often required...

DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase

International Nuclear Information System (INIS)

Strike, P.

1977-01-01

Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step. (orig.) [de

Evidence for transfer of radicals between oil-in-water emulsion droplets as detected by the probe (E,E)-3,5-Bis(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY665/676

DEFF Research Database (Denmark)

Raudsepp, Piret; Brüggemann, Dagmar Adeline; Andersen, Mogens Larsen

2014-01-01

(E,E)-3,5-Bis(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY(665/676), is a lipophilic radical-sensitive fluorescent probe that can be used to study radical-driven lipid autoxidation. The sensitivity of BODIPY(665/676) was studied in the presence of radical initiators...... di-tert-butyl peroxide and 2,2'-azobis(2,4-dimethyl)valeronitrile (AMVN). In both cases the fluorescence of BODIPY(665/676) changed more in saturated medium-chain triglyceride oil than in linseed or sunflower oils, where the high degree of unsaturation is expected to give more pronounced radical......-derived lipid oxidation. It was suggested that BODIPY(665/676), as the only available oxidizable substance in the saturated oil, was directly attacked by radicals, resulting in high rates of probe oxidation, while in the unsaturated oils, radicals attacked either unsaturated fatty acids or BODIPY(665...

The E3 ligase UBR5 regulates gastric cancer cell growth by destabilizing the tumor suppressor GKN1

International Nuclear Information System (INIS)

Yang, Min; Jiang, Nan; Cao, Qi-wei; Ma, Mao-qiang; Sun, Qing

2016-01-01

Gastric cancer is the most common digestive malignant tumor worldwide and the underlying mechanisms are not fully understood. The E3 ligase UBR5 (also known as EDD1) is essentially involved in diverse types of cancer. Here we aimed to study the functions of UBR5 in human gastric cancer. We first analyzed the mRNA and protein levels of UBR5 in human gastric cancer tissues and the results showed that UBR5 was markedly increased in gastric cancer tissues compared with normal gastric mucosa or matched non-cancer gastric tissues. The relationship between UBR5 and survival of gastric cancer patients was analyzed and we found that high UBR5 expression was associated with poor overall and disease-free survival. We further tried to investigate the effects of UBR5 on gastric cancer cell growth in vitro and in vivo. Therefore, we knocked down UBR5 with lentivirus-mediated shRNA and found that UBR5 knockdown repressed in vitro proliferation and colony formation of gastric cancer cells AGS, MG803 and MNK1. In vivo xenograft experiment also demonstrated that UBR5 knockdown inhibited AGS growth. Finally, we explored the mechanism by which UBR5 contributed to the growth of gastric cancer cells. We found that UBR5 bound the tumor suppressor gastrokine 1 (GKN1) and increased its ubiquitination to reduce the protein stability of GKN1. GKN1 knockdown with lentivirus-mediated shRNA increased the in vitro colony formation and in vivo growth of AGS cells, and UBR5 knockdown was unable to affect the colony formation and in vivo growth of AGS cells when GKN1 was knocked down, indicating that GKN1 contributed to the effects of UBR5 in human gastric cancer cells. Taken together, UBR5 plays an essential role in gastric cancer and may be a potential diagnosis and treatment target for gastric cancer. - Highlights: • UBR5 expression is up-regulated in human gastric cancer. • UBR5 overexpression predicts poor survival. • UBR5 regulates gastric cancer growth in vitro and in vivo. �

Extreme growth failure is a common presentation of ligase IV deficiency

NARCIS (Netherlands)

Murray, J.E.; Bicknell, L.S.; Yigit, G.; Duker, A.L.; Kogelenberg, M. van; Haghayegh, S.; Wieczorek, D.; Kayserili, H.; Albert, M.H.; Wise, C.A.; Brandon, J.; Kleefstra, T.; Warris, A.; Flier, M. van der; Bamforth, J.S.; Doonanco, K.; Ades, L.; Ma, A.; Field, M.; Johnson, D.; Shackley, F.; Firth, H.; Woods, C.G.; Nurnberg, P.; Gatti, R.A.; Hurles, M.; Bober, M.B.; Wollnik, B.; Jackson, A.P.

2014-01-01

Ligase IV syndrome is a rare differential diagnosis for Nijmegen breakage syndrome owing to a shared predisposition to lympho-reticular malignancies, significant microcephaly, and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying

Cullin4B/E3-ubiquitin ligase negatively regulates β-catenin

Indian Academy of Sciences (India)

-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, -catenin is targeted to ubiquitin–proteasome-mediated degradation. Here we present the functional ...

Structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

Science.gov (United States)

Deva, Taru; Baker, Edward N; Squire, Christopher J; Smith, Clyde A

2006-12-01

The bacterial cell wall provides essential protection from the external environment and confers strength and rigidity to counteract internal osmotic pressure. Without this layer the cell would be easily ruptured and it is for this reason that biosynthetic pathways leading to the formation of peptidoglycan have for many years been a prime target for effective antibiotics. Central to this pathway are four similar ligase enzymes which add peptide groups to glycan moieties. As part of a program to better understand the structure-function relationships in these four enzymes, the crystal structure of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC) has been determined to 2.6 A resolution. The structure was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and refined to a crystallographic R factor of 0.212 (R(free) = 0.259). The enzyme has a modular multi-domain structure very similar to those of other members of the mur family of ATP-dependent amide-bond ligases. Detailed comparison of these four enzymes shows that considerable conformational changes are possible. These changes, together with the recruitment of two different N-terminal domains, allow this family of enzymes to bind a substrate which is identical at one end and at the other has the growing peptide tail which will ultimately become part of the rigid bacterial cell wall. Comparison of the E. coli and Haemophilus influenzae structures and analysis of the sequences of known MurC enzymes indicate the presence of a ;dimerization' motif in almost 50% of the MurC enzymes and points to a highly conserved loop in domain 3 that may play a key role in amino-acid ligand specificity.

Novel inhibitor of DNA ligase IV with a promising cancer therapeutic ...

Indian Academy of Sciences (India)

Home; Journals; Journal of Biosciences; Volume 39; Issue 3. Novel inhibitor of DNA ligase IV with a promising cancer therapeutic potential. Ashwin Kotnis Rita Mulherkar. Clipboards Volume 39 Issue 3 June 2014 pp 339-340. Fulltext. Click here to view fulltext PDF. Permanent link:

Structure–Activity Relationship Study Based on Autoinducing Peptide (AIP) from Dog Pathogen S. schleiferi

DEFF Research Database (Denmark)

Gless, Bengt H.; Peng, Pai; Pedersen, Katja Desiree

2017-01-01

Herein,an effective protocol for solid-phase synthesis of peptidethiolactones by concomitant ring closure and cleavage from the solidsupport is reported. The strategy was applied for mapping the importanceof the structural features in S. schleiferi AIP (5) by performing an alanine scan...

Virtual screening for potential inhibitors of bacterial MurC and MurD ligases.

Science.gov (United States)

Tomašić, Tihomir; Kovač, Andreja; Klebe, Gerhard; Blanot, Didier; Gobec, Stanislav; Kikelj, Danijel; Mašič, Lucija Peterlin

2012-03-01

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

Synthesis of (E-2,4-Dinitro-N-((2E,4E-4-phenyl-5-(pyrrolidin-1-ylpenta-2,4-dienylideneaniline

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Mostafa Fesanghari

2009-07-01

Full Text Available (E-2,4-Dinitro-N-((2E,4E-4-phenyl-5-(pyrrolidin-1-ylpenta-2,4-dienylidene aniline dye was prepared in one pot by reaction of premade N-2,4-dinitrophenyl-3-phenylpyridinium chloride (DNPPC and pyrrolidine in absolute MeOH.

Unusual AIP mutation and phenocopy in the family of a young patient with acromegalic gigantism

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Syed Ali Imran

2018-01-01

Full Text Available Early-onset acromegaly causing gigantism is often associated with aryl-hydrocarbon-interacting receptor protein (AIP mutation, especially if there is a positive family history. A15y male presented with tiredness and visual problems. He was 201 cm tall with a span of 217 cm. He had typical facial features of acromegaly, elevated IGF-1, secondary hypogonadism and a large macroadenoma. His paternal aunt had a history of acromegaly presenting at the age of 35 years. Following transsphenoidal surgery, his IGF-1 normalized and clinical symptoms improved. He was found to have a novel AIP mutation destroying the stop codon c.991T>C; p.*331R. Unexpectedly, his father and paternal aunt were negative for this mutation while his mother and older sister were unaffected carriers, suggesting that his aunt represents a phenocopy.

(E-4-Bromo-N-(2,3-dimethoxybenzylideneaniline

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Karla Fejfarová

2010-09-01

Full Text Available The title Schiff base compound, C15H14BrNO2, was prepared by the condensation of 2,3-dimethoxybenzaldehyde with 4-bromoaniline. It adopts an E configuration with respect to the C=N bond. The dihedral angle between the two aromatic rings is 56.79 (8°. Weak C—H...O and C—-H...π bonds can be found in the crystal structure.

(E-3-Methyl-6-(3-oxo-3-(3,4,5-trimethoxyphenylprop-1-en-1-yl-2(3H-benzothiazolone

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Yordanka Ivanova

2016-09-01

Full Text Available The title compound, (E-3-methyl-6-(3-oxo-3-(3,4,5-trimethoxyphenylprop-1-en-1-yl-2(3H-benzothiazolone, was synthesized by both an acid- and base-catalyzed aldol condensation of 3-methyl-6-acetyl-2(3H-benzothiazolone and 3,4,5-trimethoxyacetophenone. The structure of the target compound was confirmed using 1H-NMR, 13C-NMR, IR, MS, and elemental analysis.

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

Science.gov (United States)

Wang, Li Kai; Zhu, Hui; Shuman, Stewart

2009-03-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

Directory of Open Access Journals (Sweden)

Scott Cukras

Full Text Available Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

Science.gov (United States)

Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

2014-01-01

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

KONSUMSI MIKRONUTRIEN DAN EKSPRESI GEN eNOS3 ALEL INTRO 4a4b PADA PENDERITA HIPERTENSI ETNIK MINANGKABAU

Directory of Open Access Journals (Sweden)

Delmi Sulastri

2011-05-01

Full Text Available AbstrakGen eNOS3 varian intron 4a4b merupakan salah satu gen yang berhubungan dengan kejadian hipertensi esensial. Kemampuan Gen eNOS3 varian ini untuk mensintesis enzim nitric oxide synthase (NOS berkurang sehingga sintesis Nitric oxide (NO menurun. Senyawa NO adalah suatu senyawa vasoaktif yang menyebabkan vasodilatasi pembuluh darah.Penelitian pada subjek hipertensi dan normotensi berusia 30-65 tahun bertujuan untuk melihat pengaruh mikronutrien terhadap ekspresi gen eNOS3 alel intron 4a4b. 130 subjek, diwawancara pola makannya lalu dilakukan analisis asupan mikronutrien, gen eNOS3, serta kadar NO plasma. Data yang diperoleh dianalisis menggunakan uji T dan chi-square.Kadar NO plasma penderita hipertensi adalah 26.91 + 15.40 μM/L dan normotensi 25.79 + 15.04 μM/L. 55.5% subjek hipertensi memiliki gen eNOS3 varian intron 4b dan 69% subjek normotensi mempunyai varian intron 4a4b. Kadar NO plasma rendah, (66.7% ditemukan pada subjek hipertensi dengan varian intron 4b. Tidak terdapat hubungan bermakna asupan mikronutrien dengan kadar NO plasma pada subjek hipertensi.Hasil ini tidak mendukung pengaruh konsumsi mikronutrien terhadap kadar NO plasma pada penderita hipertensi dengan gen eNOS3 varian intron 4a4b.Kata Kunci : hipertensi esensial, gen eNOS3, kadar NO plasmaAbstracteNOS 3 gene intron 4a4b allel is one of the important genes which is related to hypertesion essensial. This gene expresses the nitric oxide synthase (NOS enzyme which regulates the synthesis of Nitric oxide (NO in human body. NO causes vasodilatation, which decreases peripheral resistant and blood pressure.A research has been done based on cross sectional study on hypertension patients and those with normal blood pressure in the range of 30-65 years old. The main of this research is to evaluate the influence of micronutrient consumption on eNOS3 gene intron 4a4b allel expression in hypertension subjects in Minangkabau ethnicity. 130 subjects interviewed about micronutrient

Synthesis, spectral and antimicrobial activity of [3-(4-chloro-phenoxy-2,4-diisopropyl-2,3,4,5-tetrahydro-1H-3l5-benzo[e][1,3,2]diazaphosphepin-3-ylidene]-alkyl-amine

Directory of Open Access Journals (Sweden)

R. Usha Nagalakshmi

2009-11-01

Full Text Available 3-(4-chlorophenoxy-2,4-diisopropyl-2,3,4,5-tetrahydro-1H-benzo[e][1,3,2]diaza-phosphepine was synthesized starting from 1,2-bis(bromomethylbenzene by reacting with 2 equimolar isopropyl amine, one equimolar PBr3 and then one equimolar 4-chlorophenol, respectively. The Staudinger reaction of the diazaphphosphine with a series of alkyl azides gave the corresponding iminophosphoranes. Antibacterial activities of the synthesized iminophosphoranes were screened

Identification of Ideal Multi-targeting Bioactive Compounds Against Mur Ligases of Enterobacter aerogenes and Its Binding Mechanism in Comparison with Chemical Inhibitors.

Science.gov (United States)

Chakkyarath, Vijina; Natarajan, Jeyakumar

2017-10-31

Enterobacter aerogenes have been reported as important opportunistic and multi-resistant bacterial pathogens for humans during the last three decades in hospital wards. The emergence of drug-resistant E. aerogenes demands the need for developing new drugs. Peptidoglycan is an important component of the cell wall of bacteria and the peptidoglycan biochemical pathway is considered as the best source of antibacterial targets. Within this pathway, four Mur ligases MurC, MurD, MurE, and MurF are responsible for the successive additions of L-alanine and suitable targets for developing novel antibacterial drugs. As an inference from this fact, we modeled the three-dimensional structure of above Mur ligases using best template structures available in PDB and analyzed its common binding features. Structural refinement and energy minimization of the predicted Mur ligases models is also being done using molecular dynamics studies. The models of Mur ligases were further investigated for in silico docking studies using bioactive plant compounds from the literature. Interestingly, these results indicate that four plant compounds Isojuripidine, Atroviolacegenin, Porrigenin B, and Nummularogenin showing better docking results in terms of binding energy and number of hydrogen bonds. All these four compounds are spirostan-based compounds with differences in side chains and the amino acid such as ASN, LYS, THR, HIS, ARG (polar) and PHE, GLY, VAL, ALA, MET (non-polar) playing active role in binding site of all four Mur ligases. Overall, in the predicted model, the four plant compounds with its binding features could pave way to design novel multi-targeted antibacterial plant-based bioactive compounds specific to Mur ligases for the treatment of Enterobacter infections.

Engineering an ATP-dependent D-Ala:D-Ala ligase for synthesizing amino acid amides from amino acids.

Science.gov (United States)

Miki, Yuta; Okazaki, Seiji; Asano, Yasuhisa

2017-05-01

We successfully engineered a new enzyme that catalyzes the formation of D-Ala amide (D-AlaNH 2 ) from D-Ala by modifying ATP-dependent D-Ala:D-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of D-Ala-D-Ala from two molecules of D-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second D-Ala of D-Ala-D-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for D-AlaNH 2 production. The S293E variant, which was selected as the best enzyme for D-AlaNH 2 production, exhibited an optimal activity at pH 9.0 and 40 °C for D-AlaNH 2 production. The apparent K m values of this variant for D-Ala and NH 3 were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of D-AlaNH 2 from 10 and 50 mM D-Ala and 3 M NH 4 Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.

Crystal structure of (E-13-{4-[(Z-2-cyano-2-(3,4,5-trimethoxyphenylethenyl]phenyl}parthenolide methanol hemisolvate

Directory of Open Access Journals (Sweden)

Narsimha Reddy Penthala

2014-10-01

Full Text Available The title compound, C33H35NO6 [systematic name: (Z-3-(4-{(E-[(E-1a,5-dimethyl-9-oxo-2,3,7,7a-tetrahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-8(1aH,6H,9H,10aH,10bH-ylidene]methyl}phenyl-2-(3,4,5-trimethoxyphenylacrylonitrile methanol hemisolvate], C33H35NO6·0.5CH3OH, was prepared by the reaction of (Z-3-(4-iodophenyl-2-(3,4,5-trimethoxyphenylacrylonitrile with parthenolide [systematic name: (E-1a,5-dimethyl-8-methylene-2,3,6,7,7a,8,10a,10b-octahydrooxireno[2′,3′:9,10]cyclodeca[1,2-b]furan-9(1aH-one] under Heck reaction conditions. The molecule is built up from fused ten-, five- (lactone and three-membered (epoxide rings with a {4-[(Z-2-cyano-2-(3,4,5-trimethoxyphenylethenyl]phenyl}methylidene group as a substituent. The 4-[(Z-2-cyano-2-(3,4,5-trimethoxyphenylethenyl]phenyl group on the parthenolide exocyclic double bond is oriented in a trans position to the lactone ring to form the E isomer. The dihedral angle between the benzene ring of the phenyl moiety and the lactone ring mean plane is 21.93 (4°.

Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

Science.gov (United States)

Shanmugam, Anusuya; Natarajan, Jeyakumar

2012-06-01

Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

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

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Kimberly A. Rickman

2015-07-01

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

Exercise induced upregulation of glutamate-cysteine ligase catalytic subunit and glutamate-cysteine ligase modifier subunit gene expression in Thoroughbred horses

Directory of Open Access Journals (Sweden)

Jeong-Woong Park

2017-05-01

Full Text Available Objective This study was performed to reveal the molecular structure and expression patterns of horse glutamate-cysteine ligase catalytic subunit (GCLC and glutamate-cysteine ligase modifier subunit (GCLM genes whose products form glutamate cysteine ligase, which were identified as differentially expressed genes in the previous study. Methods We performed bioinformatics analyses, and gene expression assay with quantitative polymerase chain reaction (qPCR for horse GCLC and GCLM genes in muscle and blood leukocytes of Thoroughbred horses Results Expression of GCLC showed the same pattern in both blood and muscle tissues after exercise. Expression of GCLC increased in the muscle and blood of Thoroughbreds, suggesting a tissue-specific regulatory mechanism for the expression of GCLC. In addition, expression of the GCLM gene increased after exercise in both the blood and muscle of Thoroughbreds. Conclusion We established the expression patterns of GCLC and GCLM in the skeletal muscle and blood of Thoroughbred horses in response to exercise. Further study is now warranted to uncover the functional importance of these genes in exercise and recovery in racehorses.

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

Science.gov (United States)

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

2014-10-01

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

DEFF Research Database (Denmark)

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate......-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin...... ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

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

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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.

Expression, crystallization and preliminary X-ray crystallographic analysis of Xoo0352, d-alanine-d-alanine ligase A, from Xanthomonas oryzae pv. oryzae

International Nuclear Information System (INIS)

Doan, Thanh Thi Ngoc; Kim, Jin-Kwang; Kim, Hyesoon; Ahn, Yeh-Jin; Kim, Jeong-Gu; Lee, Byoung-Moo; Kang, Lin-Woo

2008-01-01

Xoo0352, which encodes d-alanine-d-alanine ligase A (DdlA), from X. oryzae pv. oryzae was cloned, purified and crystallized. Preliminary X-ray crystallographic analysis of DdlA crystals was performed. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB), which is one of the most devastating diseases of rice in most rice-growing countries. d-Alanine-d-alanine ligase A (DdlA), coded by the Xoo0352 gene, was expressed, purified and crystallized. DdlA is an enzyme that is involved in d-alanine metabolism and the biosynthesis of an essential bacterial peptidoglycan precursor, in which it catalyzes the formation of d-alanyl-d-alanine from two d-alanines, and is thus an attractive antibacterial drug target against Xoo. The DdlA crystals diffracted to 2.3 Å resolution and belonged to the primitive tetragonal space group P4 3 2 1 2, with unit-cell parameters a = b = 83.0, c = 97.6 Å. There is one molecule in the asymmetric unit, with a corresponding V M of 1.88 Å 3 Da −1 and a solvent content of 34.6%. The initial structure was determined by molecular replacement using d-alanine-d-alanine ligase from Staphylococcus aureus as a template model

AIP 50th anniversary: Physics vade mecum

Science.gov (United States)

Anderson, H. L.

This compendium is intended to be of use to the wide spectrum of physicists associated with the AIP through its member societies. Twenty-two subjects broadly representative of physics as a whole are discussed. Each subeditor was charged to compile within 10 pages the most useful information, formulas, numerical data, definitions, and references most physicists would like to have at hand. The General Section is a compilation of the fundamental constants, the SI units and prefixes, conversion factors, magnitudes, basic mathematical and physics formulas, formulas useful in practical physics applications, and a list of physics data centers. The particular fields considered are: acoustics, astronomy and astrophysics, atomic collision properties, atomic spectroscopy, biological physics, cryogenics, crystallography, elementary particles, energy demand, energy supply, fluid dynamics, high polymer physics, medical physics, molecular spectroscopy and structure, nuclear physics, optics, plasma physics, rheology, solid state physics, surface physics, and thermophysics.

The Banana Fruit SINA Ubiquitin Ligase MaSINA1 Regulates the Stability of MaICE1 to be Negatively Involved in Cold Stress Response.

Science.gov (United States)

Fan, Zhong-Qi; Chen, Jian-Ye; Kuang, Jian-Fei; Lu, Wang-Jin; Shan, Wei

2017-01-01

The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis . Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi- in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

3-[(E-(4-Chlorobenzylideneamino]-1-phenylthiourea

Directory of Open Access Journals (Sweden)

Nur Nadia Dzulkifli

2011-04-01

Full Text Available In the title compound, C14H12ClN3S, the dihedral angle between the terminal benzene rings is 56.6 (2°; the benzene rings lie to the same side of the molecule. The major twist in the molecule occurs around the Car—N bond (ar is aromatic [C—N—C—C = 49.9 (5°]. The configuration about the N=C bond [1.271 (4 Å] is E. The amine H atoms lie on opposite sides of the molecule with one forming an intramolecular N—H...N(imine hydrogen bond and an S(5 ring. In the crystal, centrosymmetric dimers are formed via {...HNC=S}2 synthons.

Advanced Information Processing System (AIPS)-based fault tolerant avionics architecture for launch vehicles

Science.gov (United States)

Lala, Jaynarayan H.; Harper, Richard E.; Jaskowiak, Kenneth R.; Rosch, Gene; Alger, Linda S.; Schor, Andrei L.

1990-01-01

An avionics architecture for the advanced launch system (ALS) that uses validated hardware and software building blocks developed under the advanced information processing system program is presented. The AIPS for ALS architecture defined is preliminary, and reliability requirements can be met by the AIPS hardware and software building blocks that are built using the state-of-the-art technology available in the 1992-93 time frame. The level of detail in the architecture definition reflects the level of detail available in the ALS requirements. As the avionics requirements are refined, the architecture can also be refined and defined in greater detail with the help of analysis and simulation tools. A useful methodology is demonstrated for investigating the impact of the avionics suite to the recurring cost of the ALS. It is shown that allowing the vehicle to launch with selected detected failures can potentially reduce the recurring launch costs. A comparative analysis shows that validated fault-tolerant avionics built out of Class B parts can result in lower life-cycle-cost in comparison to simplex avionics built out of Class S parts or other redundant architectures.

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

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

2016-01-01

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

Aggressive pituitary adenomas occurring in young patients in a large Polynesian kindred with a germline R271W mutation in the AIP gene.

OpenAIRE

Jennings, J. E.; Georgitsi, M.; Holdaway, I.; Daly, Adrian; Tichomirowa, M.; Beckers, Albert; Aaltonen, Lauri A; Karhu, A.; Cameron, F. J.

2009-01-01

OBJECTIVE: Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) were recently shown to confer a pituitary adenoma predisposition in patients with familial isolated pituitary adenomas (FIPA). We report a large Samoan FIPA kindred from Australia/New Zealand with an R271W mutation that was associated with aggressive pituitary tumors. DESIGN AND METHODS: Case series with germline screening of AIP and haplotype analyses among R271W families. RESULTS: This previously unreported kind...

Is e+e- pair emission important in the determination of the 3He+4He S factor?

Science.gov (United States)

Snover, K. A.; Hurd, A. E.

2003-05-01

We show that the cross section for direct E0 pair emission is related to the cross section for direct E2 photon emission, and is a negligible contribution to the total capture cross section for 3He+4He→7Be. E0 resonance emission, E1 pair emission, and internal conversion are also negligible. Thus there cannot be significant contributions to the 3He+4He→7Be capture cross section at low energies from electromagnetic emission processes other than single photon emission.

Hypermethylation of MST1 in IgG4-related autoimmune pancreatitis and rheumatoid arthritis

Energy Technology Data Exchange (ETDEWEB)

Fukuhara, Takataro; Tomiyama, Takashi [Division of Gastroenterology and Hepatology, The Third Department of Internal Medicine, JST CREST, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan); Yasuda, Kaneki [Department of Urology and Andrology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan); Ueda, Yoshihiro [Department of Molecular Genetics, Institute of Biomedical Science, and JST CREST, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan); Ozaki, Yoshio; Son, Yonsu; Nomura, Shosaku [Department of the First Department of Internal Medicine, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan); Uchida, Kazushige; Okazaki, Kazuichi [Division of Gastroenterology and Hepatology, The Third Department of Internal Medicine, JST CREST, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan); Kinashi, Tatsuo, E-mail: kinashi@takii.kmu.ac.jp [Department of Molecular Genetics, Institute of Biomedical Science, and JST CREST, Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka 573-1010 (Japan)

2015-08-07

The serine/threonine kinase Mst1 plays important roles in the control of immune cell trafficking, proliferation, and differentiation. Previously, we reported that Mst1 was required for thymocyte selection and regulatory T-cell functions, thereby the prevention of autoimmunity in mice. In humans, MST1 null mutations cause T-cell immunodeficiency and hypergammaglobulinemia with autoantibody production. RASSF5C(RAPL) is an activator of MST1 and it is frequently methylated in some tumors. Herein, we investigated methylation of the promoter regions of MST1 and RASSF5C(RAPL) in leukocytes from patients with IgG4-related autoimmune pancreatitis (AIP) and rheumatoid arthritis (RA). Increased number of CpG methylation in the 5′ region of MST1 was detected in AIP patients with extrapancreatic lesions, whereas AIP patients without extrapancreatic lesions were similar to controls. In RA patients, we detected a slight increased CpG methylation in MST1, although the overall number of methylation sites was lower than that of AIP patients with extrapancreatic lesions. There were no significant changes of the methylation levels of the CpG islands in the 5′ region of RASSF5C(RAPL) in leukocytes from AIP and RA patients. Consistently, we found a significantly down-regulated expression of MST1 in regulatory T cells of AIP patients. Our results suggest that the decreased expression of MST1 in regulatory T cells due to hypermethylation of the promoter contributes to the pathogenesis of IgG4-related AIP. - Highlights: • Mst1 controls immune cells trafficking, cell proliferation and differentiation. • Autoimmune pancreatitis (AIP) is an idiopathic pancreatitis affecting multiple organs. • Decreased MST1 expression and increased CpG methylation of promoter of MST1 in AIP. • Slight increased CpG methylation of MST1 in rheumatoid arthritis patients. • MST1 contributes pathogenesis of IgG4-related AIP.

Hypermethylation of MST1 in IgG4-related autoimmune pancreatitis and rheumatoid arthritis

International Nuclear Information System (INIS)

Fukuhara, Takataro; Tomiyama, Takashi; Yasuda, Kaneki; Ueda, Yoshihiro; Ozaki, Yoshio; Son, Yonsu; Nomura, Shosaku; Uchida, Kazushige; Okazaki, Kazuichi; Kinashi, Tatsuo

2015-01-01

The serine/threonine kinase Mst1 plays important roles in the control of immune cell trafficking, proliferation, and differentiation. Previously, we reported that Mst1 was required for thymocyte selection and regulatory T-cell functions, thereby the prevention of autoimmunity in mice. In humans, MST1 null mutations cause T-cell immunodeficiency and hypergammaglobulinemia with autoantibody production. RASSF5C(RAPL) is an activator of MST1 and it is frequently methylated in some tumors. Herein, we investigated methylation of the promoter regions of MST1 and RASSF5C(RAPL) in leukocytes from patients with IgG4-related autoimmune pancreatitis (AIP) and rheumatoid arthritis (RA). Increased number of CpG methylation in the 5′ region of MST1 was detected in AIP patients with extrapancreatic lesions, whereas AIP patients without extrapancreatic lesions were similar to controls. In RA patients, we detected a slight increased CpG methylation in MST1, although the overall number of methylation sites was lower than that of AIP patients with extrapancreatic lesions. There were no significant changes of the methylation levels of the CpG islands in the 5′ region of RASSF5C(RAPL) in leukocytes from AIP and RA patients. Consistently, we found a significantly down-regulated expression of MST1 in regulatory T cells of AIP patients. Our results suggest that the decreased expression of MST1 in regulatory T cells due to hypermethylation of the promoter contributes to the pathogenesis of IgG4-related AIP. - Highlights: • Mst1 controls immune cells trafficking, cell proliferation and differentiation. • Autoimmune pancreatitis (AIP) is an idiopathic pancreatitis affecting multiple organs. • Decreased MST1 expression and increased CpG methylation of promoter of MST1 in AIP. • Slight increased CpG methylation of MST1 in rheumatoid arthritis patients. • MST1 contributes pathogenesis of IgG4-related AIP

RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation.

Science.gov (United States)

Azmi, Peter; Seth, Arun

2005-11-01

Our laboratory has found that the 154aa RING finger protein 11 (RNF11), has modular domains and motifs including a RING-H2 finger domain, a PY motif, an ubiquitin interacting motif (UIM), a 14-3-3 binding sequence and an AKT phosphorylation site. RNF11 represents a unique protein with no other known immediate family members yet described. Comparative genetic analysis has shown that RNF11 is highly conserved throughout evolution. This may indicate a conserved and non-redundant role for the RNF11 protein. Molecular binding assays using RNF11 have shown that RNF11 has important roles in growth factor signalling, ubiquitination and transcriptional regulation. RNF11 has been shown to interact with HECT-type E3 ubiquitin ligases Nedd4, AIP4, Smurf1 and Smurf2, as well as with Cullin1, the core protein in the multi-subunit SCF E3 ubiquitin ligase complex. Work done in our laboratory has shown that RNF11 is capable of antagonizing Smurf2-mediated inhibition of TGFbeta signalling. Furthermore, RNF11 is capable of degrading AMSH, a positive regulator of both TGFbeta and EGFR signalling pathways. Recently, we have found that RNF11 can directly enhance TGFbeta signalling through a direct association with Smad4, the common signal transducer and transcription factor in the TGFbeta, BMP, and Activin pathways. Through its association with Smad4 and other transcription factors, RNF11 may have a role in direct transcriptional regulation. Our laboratory and others have found nearly 80 protein interactions for RNF11, placing RNF11 at the cross-roads of cell signalling and transcriptional regulation. RNF11 is highly expressed in breast tumours. Deregulation of RNF11 function may prove to be harmful to patient therapeutic outcomes. RNF11 may therefore provide a novel target for cancer therapeutics. The purpose of this review is to discuss the role of RNF11 in cell signalling and transcription factor modulation with special attention given to the ubiquitin-proteasomal pathway, TGFbeta

The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

Science.gov (United States)

Feng, Youjun; Chin, Chui-Yoke; Chakravartty, Vandana; Gao, Rongsui; Crispell, Emily K.

2015-01-01

ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. PMID:26060274

Probing ligand binding modes of Mycobacterium tuberculosis MurC ligase by molecular modeling, dynamics simulation and docking.

Science.gov (United States)

Anuradha, C M; Mulakayala, Chaitanya; Babajan, Banaganapalli; Naveen, M; Rajasekhar, Chikati; Kumar, Chitta Suresh

2010-01-01

Multi drug resistance capacity for Mycobacterium tuberculosis (MDR-Mtb) demands the profound need for developing new anti-tuberculosis drugs. The present work is on Mtb-MurC ligase, which is an enzyme involved in biosynthesis of peptidoglycan, a component of Mtb cell wall. In this paper the 3-D structure of Mtb-MurC has been constructed using the templates 1GQQ and 1P31. Structural refinement and energy minimization of the predicted Mtb-MurC ligase model has been carried out by molecular dynamics. The streochemical check failures in the energy minimized model have been evaluated through Procheck, Whatif ProSA, and Verify 3D. Further torsion angles for the side chains of amino acid residues of the developed model were determined using Predictor. Docking analysis of Mtb-MurC model with ligands and natural substrates enabled us to identify specific residues viz. Gly125, Lys126, Arg331, and Arg332, within the Mtb-MurC binding pocket to play an important role in ligand and substrate binding affinity and selectivity. The availability of Mtb-MurC ligase built model, together with insights gained from docking analysis will promote the rational design of potent and selective Mtb-MurC ligase inhibitors as antituberculosis therapeutics.

AcEST: DK954361 [AcEST

Lifescience Database Archive (English)

Full Text Available in 5-4 OS=Homo sap... 33 1.1 sp|Q9DBY1|SYVN1_MOUSE E3 ubiquitin-protein ligase synoviolin OS=... 33 1.4 sp|Q...86TM6|SYVN1_HUMAN E3 ubiquitin-protein ligase synoviolin OS=... 33 1.4 sp|O55188|DMP1_MOUSE Dentin matrix ac

Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

Science.gov (United States)

Kadokura, M; Wada, T; Seio, K; Sekine, M

2000-08-25

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

DNA ligase III is involved in a DNA-PK independent pathway of NHEJ in human cells

International Nuclear Information System (INIS)

Wang, H.; Perrault, A.R.; Qin, W.; Wang, H.; Iliakis, G.

2003-01-01

Full text: Double strand breaks (DSB) induced by ionizing radiation (IR) and other cytotoxic agents in the genome of higher eukaryotes are thought to be repaired either by homologous recombination repair (HRR), or non-homologous endjoining (NHEJ). We previously reported the operation of two components of NHEJ in vivo: a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK independent component that acts as a backup (basic or B-NHEJ) and operates with kinetics an order of magnitude slower. To gain further insight into the mechanisms of B-NHEJ, we investigated DNA endjoining in extracts 180BR, a human cell line deficient in DNA ligase IV, using an in vitro plasmid-based DNA endjoining assay. An anti DNA ligase III antibody inhibited almost completely DNA endjoining activity in these extracts. On the other hand, an anti DNA ligase I antibody had no measurable effect in DNA endjoining activity. Immunodepletion of DNA ligase III from 180BR cell extracts abolished the DNA endjoining activity, which could be restored by addition of purified human DNA ligase IIIb. Full-length DNA ligase III bound to double stranded DNA and stimulated DNA endjoining in both intermolecular and intramolecular ligation. Furthermore, fractionation of HeLa cell extracts demonstrated the presence of an activity stimulating the function of DNA ligase III. Based on these observations we propose that DNA ligase III is the ligase operating in B-NHEJ

Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

DEFF Research Database (Denmark)

Andresen, Christina Aaen; Smedegaard, Stine; Sylvestersen, Kathrine Beck

2014-01-01

The Ankyrin and SOCS (Suppressor of Cytokine Signaling) box (ASB) family of proteins function as the substrate recognition subunit in a subset of Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligases. Despite counting with 18 members in humans, the identity of the physiological targets of the Asb protei...

Systematic Analysis of the 4-Coumarate:Coenzyme A Ligase (4CL Related Genes and Expression Profiling during Fruit Development in the Chinese Pear

Directory of Open Access Journals (Sweden)

Yunpeng Cao

2016-10-01

Full Text Available In plants, 4-coumarate:coenzyme A ligases (4CLs, comprising some of the adenylate-forming enzymes, are key enzymes involved in regulating lignin metabolism and the biosynthesis of flavonoids and other secondary metabolites. Although several 4CL-related proteins were shown to play roles in secondary metabolism, no comprehensive study on 4CL-related genes in the pear and other Rosaceae species has been reported. In this study, we identified 4CL-related genes in the apple, peach, yangmei, and pear genomes using DNATOOLS software and inferred their evolutionary relationships using phylogenetic analysis, collinearity analysis, conserved motif analysis, and structure analysis. A total of 149 4CL-related genes in four Rosaceous species (pear, apple, peach, and yangmei were identified, with 30 members in the pear. We explored the functions of several 4CL and acyl-coenzyme A synthetase (ACS genes during the development of pear fruit by quantitative real-time PCR (qRT-PCR. We found that duplication events had occurred in the 30 4CL-related genes in the pear. These duplicated 4CL-related genes are distributed unevenly across all pear chromosomes except chromosomes 4, 8, 11, and 12. The results of this study provide a basis for further investigation of both the functions and evolutionary history of 4CL-related genes.

The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax

Science.gov (United States)

Fryrear, Kimberly A.; Guo, Xin

2012-01-01

The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)–modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB–mediated and decreased cAMP Response Element-Binding (CREB)–mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage–induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes. PMID:22106342

A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

Science.gov (United States)

Lohman, Gregory J S; Bauer, Robert J; Nichols, Nicole M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Evans, Thomas C

2016-01-29

DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

Directory of Open Access Journals (Sweden)

Jan Mareš

Full Text Available A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea. Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

The Cytoprotective Effects of E-α-(4-Methoxyphenyl-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC--A Novel and Non-Cytotoxic HO-1 Inducer.

Directory of Open Access Journals (Sweden)

Kai B Kaufmann

Full Text Available Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1, is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264

Application of an Acyl-CoA Ligase from Streptomyces aizunensis for Lactam Biosynthesis

DEFF Research Database (Denmark)

Zhang, Jingwei; Barajas, Jesus F.; Burdu, Mehmet

2017-01-01

lactams under ambient conditions. In this study, we demonstrated production of these chemicals using ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-02301 in Streptomyces aizunensis. This enzyme has a broad substrate spectrum and can cyclize 4-aminobutyric acid into γ-butyrolactam, 5...

MAGE-A Cancer/Testis Antigens Inhibit MDM2 Ubiquitylation Function and Promote Increased Levels of MDM4

OpenAIRE

Marcar, Lynnette; Ihrig, Bianca; Hourihan, John; Bray, Susan E; Quinlan, Philip R; Jordan, Lee B; Thompson, Alastair M; Hupp, Ted R; Meek, David W

2015-01-01

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquityla...

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

Science.gov (United States)

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

2015-07-07

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Schmidt Reaction of E-3-Benzylidenechromanones and E-3-Benzylidenethiochromanones

Directory of Open Access Journals (Sweden)

Tapas K. Mandal

2013-01-01

Full Text Available On treatment with NaN3/c. H2SO4-HOAc or NaN3/TFA, E-3-benzylidenechromanones are mostly converted to E-β-aminobenzylidenechromanones while E-3-benzylidenethiochromanones are converted to 3-benzoylthiochromones. A structurally new type of product has been isolated for the reaction of E-3-benzylidene-4′-methoxychromanone with NaN3/TFA. Mechanistic paths have been suggested for formation of the products.

HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.

Science.gov (United States)

Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J N Mark; Giam, Chou-Zen

2015-08-01

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

AcEST: DK957523 [AcEST

Lifescience Database Archive (English)

Full Text Available th CABYR-binding protein OS=Mus... 33 1.1 sp|Q9DBY1|SYVN1_MOUSE E3 ubiquitin-protein ligase synoviolin OS=...... 33 1.4 sp|Q86TM6|SYVN1_HUMAN E3 ubiquitin-protein ligase synoviolin OS=... 33 1.4 sp|O55188|DMP1_MOUSE Den

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression.

Science.gov (United States)

David, Diana; Surendran, Arun; Thulaseedharan, Jissa V; Nair, Asha S

2018-03-13

Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation. CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines. Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf

Effects of Δ-isobar degrees of freedom on the reactions 3He(n,γ)4He and 3He(p,e+νe)4He at low-energy

International Nuclear Information System (INIS)

Schiavilla, R.

1991-01-01

The cross sections of the radiative 3 He(n,γ) 4 He and weak 3 He(p,e + ν e ) 4 He capture reactions at thermal neutron and keV proton energies have been calculated with the Variational Monte Carlo method. The ground state and low-energy continuum wave functions have been determined variationally from a realistic Hamiltonian, and include both nucleon and Δ-isobar degrees of freedom. The electroweak transition operator contains one- and two-body components in the N + Δ Hilbert space

Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains.

Science.gov (United States)

Yang, S W; Becker, F F; Chan, J Y

1990-10-25

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel

AIP – Aterogenní index plazmy jako významný prediktor kardiovaskulárního rizika: od výzkumu do praxe

Czech Academy of Sciences Publication Activity Database

Dobiášová, Milada

2006-01-01

Roč. 52, č. 1 (2006), s. 64-71 ISSN 0042-773X. [Pokroky ve farmakoterapii 2005 pro lékaře v praxi. Zlín, 07.09.2005-09.09.2005] R&D Projects: GA MZd(CZ) NR8328; GA MZd(CZ) NA6590; GA ČR(CZ) GV306/96/K220 Institutional research plan: CEZ:AV0Z50110509 Keywords : AIP * Log (TG/HDL-C) * particle size of lipoproteins Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

Protein: FBA3 [TP Atlas

Lifescience Database Archive (English)

Full Text Available FBA3 Ubiquitination CBLB RNF56 CBLB E3 ubiquitin-protein ligase CBL-B Casitas B-lineage lymphoma pr...oto-oncogene b, RING finger protein 56, SH3-binding protein CBL-B, Signal transduction prote

School Board Improvement Plans in Relation to the AIP Model of Educational Accountability: A Content Analysis

Science.gov (United States)

van Barneveld, Christina; Stienstra, Wendy; Stewart, Sandra

2006-01-01

For this study we analyzed the content of school board improvement plans in relation to the Achievement-Indicators-Policy (AIP) model of educational accountability (Nagy, Demeris, & van Barneveld, 2000). We identified areas of congruence and incongruence between the plans and the model. Results suggested that the content of the improvement…

ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

DEFF Research Database (Denmark)

Zhao, A.; Gray, F. C; MacNeill, S. A.

2006-01-01

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

The Role of the CRL4Cdt2 Target Spd1 in Chromosome Segregation in Fission Yeast

DEFF Research Database (Denmark)

Landvad, Katrine

Ddb1, a component of the E3 ubiquitin ligase CRL4Cdt2, is needed for proper chromosome segregation in fission yeast as ddb1 deleted cells show unequal distribution of DNA to daughter cells and sensitivity to the microtubule destabilising drug TBZ. In this study we show that Δddb1 cells have...

Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

Energy Technology Data Exchange (ETDEWEB)

Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

2015-06-12

XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.

Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

Science.gov (United States)

Ehmann, David E; Demeritt, Julie E; Hull, Kenneth G; Fisher, Stewart L

2004-05-06

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.

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness.

Directory of Open Access Journals (Sweden)

Nicole Brimer

2017-12-01

Full Text Available Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A, by binding to an LXXLL peptide (ELTLQELLGEE displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs and cetacean (porpoises and dolphins hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution.

Siah1/2 Ubiquitin Ligases in ER Stress Signaling in Melanoma

Science.gov (United States)

2016-12-01

enriched (and downregulated) upon Siah2 KD (Fig 4). RNAseq to identify genes and pathways that are deregulated in the Siah2 KD melanomas, led us...June 2016), Signgene Symposium (Berlin, Germany ; Sept. 2016), European Society for Pigment Cell Research (Milano, Italy; Sept. 2016), Centre National...Flaherty KT, Ronai ZA. Downregulation of the Ubiquitin Ligase RNF125 Underlies Resistance of Melanoma Cells to BRAF Inhibitors via JAK1 Deregulation

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

Science.gov (United States)

Lian, Hong; Zeldes, Benjamin M; Lipscomb, Gina L; Hawkins, Aaron B; Han, Yejun; Loder, Andrew J; Nishiyama, Declan; Adams, Michael W W; Kelly, Robert M

2016-12-01

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 - and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO 2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO 2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO 2 into 3HP production in metabolically engineered P

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

Science.gov (United States)

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

2008-07-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

LENUS (Irish Health Repository)

O'Connor, T M

2012-02-03

BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1976-01-01

Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis (''exaggerated'' repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5' → 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis, but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action

Light Distribution in the E3 and E4 Scintillation Counters of the ATLAS Tile Calorimeter

CERN Document Server

Hsu, Catherine

2013-01-01

The Tile Calorimeter (TileCal) of the ATLAS experiment is an important component of the ATLAS calorimetry because they play a crucial role in the search for new particles. The E3 and E4 are crack scintillators of TileCal that extend into the gap region between the EM barrel and EM endcaps. They thus sample the energy of the EM showers produced by particles interacting with the dead material in the EM calorimeters and with the inner detector cables. This project focuses on the study of the light collection uniformity in the E3 and E4 scintillating tiles using low energy electrons as the ionising particles. It is important to have uniform light response in the tiles because it would ensure a good energy resolution for the dead region. However, many factors affect the uniform light collection within the scintillating tiles.

SU-F-R-52: A Comparison of the Performance of Radiomic Features From Free Breathing and 4DCT Scans in Predicting Disease Recurrence in Lung Cancer SBRT Patients

International Nuclear Information System (INIS)

Huynh, E; Coroller, T; Narayan, V; Agrawal, V; Romano, J; Franco, I; Hou, Y; Mak, R; Aerts, H

2016-01-01

Purpose: There is a clinical need to identify patients who are at highest risk of recurrence after being treated with stereotactic body radiation therapy (SBRT). Radiomics offers a non-invasive approach by extracting quantitative features from medical images based on tumor phenotype that is predictive of an outcome. Lung cancer patients treated with SBRT routinely undergo free breathing (FB image) and 4DCT (average intensity projection (AIP) image) scans for treatment planning to account for organ motion. The aim of the current study is to evaluate and compare the prognostic performance of radiomic features extracted from FB and AIP images in lung cancer patients treated with SBRT to identify which image type would generate an optimal predictive model for recurrence. Methods: FB and AIP images of 113 Stage I-II NSCLC patients treated with SBRT were analysed. The prognostic performance of radiomic features for distant metastasis (DM) was evaluated by their concordance index (CI). Radiomic features were compared with conventional imaging metrics (e.g. diameter). All p-values were corrected for multiple testing using the false discovery rate. Results: All patients received SBRT and 20.4% of patients developed DM. From each image type (FB or AIP), nineteen radiomic features were selected based on stability and variance. Both image types had five common and fourteen different radiomic features. One FB (CI=0.70) and five AIP (CI range=0.65–0.68) radiomic features were significantly prognostic for DM (p<0.05). None of the conventional features derived from FB images (range CI=0.60–0.61) were significant but all AIP conventional features were (range CI=0.64–0.66). Conclusion: Features extracted from different types of CT scans have varying prognostic performances. AIP images contain more prognostic radiomic features for DM than FB images. These methods can provide personalized medicine approaches at low cost, as FB and AIP data are readily available within a large

SU-F-R-52: A Comparison of the Performance of Radiomic Features From Free Breathing and 4DCT Scans in Predicting Disease Recurrence in Lung Cancer SBRT Patients

Energy Technology Data Exchange (ETDEWEB)

Huynh, E; Coroller, T; Narayan, V; Agrawal, V; Romano, J; Franco, I; Hou, Y; Mak, R; Aerts, H [Dana-Farber Cancer Institute, Brigham Women’s Hospital, Harvard Medical School, Boston, MA (United States)

2016-06-15

Purpose: There is a clinical need to identify patients who are at highest risk of recurrence after being treated with stereotactic body radiation therapy (SBRT). Radiomics offers a non-invasive approach by extracting quantitative features from medical images based on tumor phenotype that is predictive of an outcome. Lung cancer patients treated with SBRT routinely undergo free breathing (FB image) and 4DCT (average intensity projection (AIP) image) scans for treatment planning to account for organ motion. The aim of the current study is to evaluate and compare the prognostic performance of radiomic features extracted from FB and AIP images in lung cancer patients treated with SBRT to identify which image type would generate an optimal predictive model for recurrence. Methods: FB and AIP images of 113 Stage I-II NSCLC patients treated with SBRT were analysed. The prognostic performance of radiomic features for distant metastasis (DM) was evaluated by their concordance index (CI). Radiomic features were compared with conventional imaging metrics (e.g. diameter). All p-values were corrected for multiple testing using the false discovery rate. Results: All patients received SBRT and 20.4% of patients developed DM. From each image type (FB or AIP), nineteen radiomic features were selected based on stability and variance. Both image types had five common and fourteen different radiomic features. One FB (CI=0.70) and five AIP (CI range=0.65–0.68) radiomic features were significantly prognostic for DM (p<0.05). None of the conventional features derived from FB images (range CI=0.60–0.61) were significant but all AIP conventional features were (range CI=0.64–0.66). Conclusion: Features extracted from different types of CT scans have varying prognostic performances. AIP images contain more prognostic radiomic features for DM than FB images. These methods can provide personalized medicine approaches at low cost, as FB and AIP data are readily available within a large

Características de carcaça e da carne de novilhas Charolês e 3/4 Charolês 1/4 Nelore, terminadas em confinamento Carcass and meat characteristics of Charolais and 3/4 Charolais 1/4 Nellore cull heifers, finished in feedlot

Directory of Open Access Journals (Sweden)

João Restle

2001-06-01

Full Text Available O objetivo deste trabalho foi estudar os aspectos quantitativos e qualitativos da carcaça e da carne de novilhas de descarte dos genótipos Charolês (C e 3/4 C 1/4 Nelore (N. Foram usadas 25 novilhas de três anos, terminadas em confinamento por 80 dias. Não se verificou diferença para peso de abate (473,6 vs. 468,8 kg, peso de carcaça fria (241,8 vs. 241,8 kg, rendimento de carcaça fria (51,09 vs. 51,61%, espessura de gordura (4,13 vs. 4,78 mm, porcentagem de dianteiro (35,52 vs. 36,01% e de costilhar (15,03 vs. 15,42%, conformação (10,75 vs. 10,56 pontos e comprimento de carcaça (127,4 vs. 129,9 cm. As novilhas C apresentaram maior porcentagem do corte serrote (49,95 vs. 48,57% e perímetro de braço (36,73 vs. 35,00 cm que as ¾ C ¼ N. Não houve diferença significativa para a composição física da carcaça, cor (3,46 vs. 3,11 pontos, textura (3,67 vs. 3,33 pontos, marmoreio (6,40 vs. 5,00 pontos, maciez (6,48 vs. 6,19 pontos, palatabilidade (6,33 vs. 6,33 pontos e força para corte das fibras da carne, por meio do aparelho WB-Shear (5,99 vs. 6,24 pontos. A suculência da carne foi melhor nas novilhas C (6,18 vs. 5,67 pontos. A partir desses resultados, pode-se concluir que, para novilhas abatidas aos três anos, não existem diferenças expressivas nos aspectos quantitativos e qualitativos da carcaça e da carne entre os dois genótipos.The objective of this work was to study the quantitative and qualitative carcass and meat characteristics of Charolais (C and 3/4 C 1/4 Nellore (N cull heifers. Twenty-five culled heifers, three years old, were feedlot finished per 80 days period. No significant difference was observed for slaughter weight (473.6 vs. 468.8 kg, cold carcass weight (241.8 vs. 241.8 kg, dressing percentage (51.09 vs. 51.61%, fat thickness (4.13 vs. 4.78 mm, forequarter percentage (35.52 vs. 36.01%, sidecut percentage (15.03 vs. 15.42%, conformation (10.75 vs. 10.56 points and carcass length (127.4 vs. 129.9 cm. The

G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

International Nuclear Information System (INIS)

Brooks, William S.; Banerjee, Sami; Crawford, David F.

2007-01-01

G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

Science.gov (United States)

Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

OpenAIRE

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D.; Pelletier, Jerry; Ferraiuolo, Maria A.; Sonenberg, Nahum

2008-01-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported...

High-temperature stability of thermoelectric Ca₃Co₄O₉ thin films

DEFF Research Database (Denmark)

Brinks, P.; Van Nong, Ngo; Pryds, Nini

2015-01-01

An enhanced thermal stability in thermoelectric Ca3Co4O9 thin films up to 550 °C in an oxygen rich environment was demonstrated by high-temperature electrical and X-ray diffraction measurements. In contrast to generally performed heating in helium gas, it is shown that an oxygen/helium mixture...... provides sufficient thermal contact, while preventing the previously disregarded formation of oxygen vacancies. Combining thermal cycling with electrical measurements proves to be a powerful tool to study the real intrinsic thermoelectric behaviour of oxide thin films at elevated temperatures. © 2015 AIP...

Finding the 3-gluon vertex from 4-jet events in e+e- annihilation