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Sample records for 10-kilodalton acyl-coenzyme a-binding

  1. Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

    USDA-ARS?s Scientific Manuscript database

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

  2. Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells*s⃞

    PubMed Central

    Soupene, Eric; Serikov, Vladimir; Kuypers, Frans A.

    2008-01-01

    Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development. PMID:18268358

  3. Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds1[C][W][OPEN

    PubMed Central

    Yurchenko, Olga; Singer, Stacy D.; Nykiforuk, Cory L.; Gidda, Satinder; Mullen, Robert T.; Moloney, Maurice M.; Weselake, Randall J.

    2014-01-01

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. PMID:24740000

  4. Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

    PubMed

    Yurchenko, Olga; Singer, Stacy D; Nykiforuk, Cory L; Gidda, Satinder; Mullen, Robert T; Moloney, Maurice M; Weselake, Randall J

    2014-06-01

    Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1(cisΔ11)) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2(cisΔ9,12); 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1(cisΔ11) (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3(cisΔ9,12,15)) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

  5. Acyl-Coenzyme A Binding Protein Regulates Beta Oxidation Required for Growth and Survival of Non-Small Cell Lung Cancer

    PubMed Central

    Harris, Fredrick T.; Rahman, S.M. Jamshedur; Hassanein, Mohamed; Qian, Jun; Hoeksema, Megan D.; Chen, Heidi; Eisenberg, Rosana; Chaurand, Pierre; Caprioli, Richard M.; Shiota, Masakazu; Massion, Pierre P.

    2014-01-01

    We identified Acyl-Coenzyme A Binding Protein (ACBP) as part of a proteomic signature predicting the risk of having lung cancer. Because ACBP is known to regulate beta oxidation (β-oxidation), which in turn controls cellular proliferation, we hypothesized that ACBP contributes to regulation of cellular proliferation and survival of non-small cell lung cancer (NSCLC) by modulating β-oxidation. We utilized matrix assisted laser desorption ionization- imaging mass spectrometry (MALDI-IMS) and immunohistochemistry (IHC) to confirm ACBP’s tissue localization in pre-invasive and invasive NSCLCs. We correlated ACBP gene expression levels in NSCLC with clinical outcomes. In loss of function studies, we tested the effect of the downregulation of ACBP on cellular proliferation and apoptosis in normal bronchial and NSCLC cell lines. Using tritiated-palmitate (3H-palmitate), we measured β-oxidation levels and tested the effect of etomoxir, a β-oxidation inhibitor, on proliferation and apoptosis. MALDI-IMS and IHC analysis confirmed that ACBP is overexpressed in preinvasive and invasive lung cancers. High ACBP gene expression levels in NSCLCs correlated with worse survival (HR = 1.73). We observed a 40% decrease in β-oxidation and concordant decreases in proliferation and increases in apoptosis in ACBP depleted NSCLC cells as compared to bronchial airway epithelial cells. Inhibition of β-oxidation by etomoxir in ACBP overexpressing cells produced dose-dependent decrease in proliferation, and increase in apoptosis (p=0.01 and p <0.001 respectively). These data suggest a role for ACBP in controlling lung cancer progression by regulating β-oxidation. PMID:24819876

  6. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease. PMID:19141679

  7. Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25

    SciTech Connect

    Chang, C.C.Y.; Chang, W.; Chang, T.Y. ); Noll, W.W.; Nutile-McMenemy, N. ); Lindsay, E.A.; Baldini, A. )

    1994-01-01

    Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently the authors' laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. They have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human-hamster somatic cell hybrid panels.

  8. MRI in medium-chain acyl-coenzyme a dehydrogenase deficiency: neuroimaging during the first month.

    PubMed

    Talamanca, Lorenzo Figà; Pasquini, Luca; Napolitano, Antonio; Longo, Daniela

    2017-08-28

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) is the most common genetic disorder of fatty acid oxidation, which presents before the age of 2 with the onset of acute hypoketotic hypoglycemia, and is typically precipitated by stress. We report serial brain magnetic resonance imaging (MRI) changes, including MR spectroscopy (MRS) and diffusion weighted imaging (DWI), in a patient with a classical MCAD presentation, compared with five healthy controls. Through this unique case we analyze the evolution of radiological findings during the first month of illness and we highlight the pivotal role of MRI, especially DWI, in the early diagnosis of the decompensated state of the disease.

  9. Purification and Characterization of a Novel Pumpkin Short-Chain Acyl-Coenzyme A Oxidase with Structural Similarity to Acyl-Coenzyme A Dehydrogenases

    PubMed Central

    De Bellis, Luigi; Gonzali, Silvia; Alpi, Amedeo; Hayashi, Hiroshi; Hayashi, Makoto; Nishimura, Mikio

    2000-01-01

    A novel pumpkin (Cucurbita pepo) short-chain acyl-coenzyme A (CoA) oxidase (ACOX) was purified to homogeneity by hydrophobic-interaction, hydroxyapatite, affinity, and anion-exchange chromatography. The purified enzyme is a tetrameric protein, consisting of apparently identical 47-kD subunits. The protein structure of this oxidase differs from other plant and mammalian ACOXs, but is similar to the protein structure of mammalian mitochondrial acyl-CoA dehydrogenase (ACDH) and the recently identified plant mitochondrial ACDH. Subcellular organelle separation by sucrose density gradient centrifugation revealed that the enzyme is localized in glyoxysomes, whereas no immunoreactive bands of similar molecular weight were detected in mitochondrial fractions. The enzyme selectively catalyzes the oxidation of CoA esters of fatty acids with 4 to 10 carbon atoms, and exhibits the highest activity on C-6 fatty acids. Apparently, the enzyme has no activity on CoA esters of branched-chain or dicarboxylic fatty acids. The enzyme is slightly inhibited by high concentrations of substrate and it is not inhibited by Triton X-100 at concentrations up to 0.5% (v/v). The characteristics of this novel ACOX enzyme are discussed in relation to other ACOXs and ACDHs. PMID:10806249

  10. Discovery of novel acyl coenzyme a: cholesterol acyltransferase inhibitors: pharmacophore-based virtual screening, synthesis and pharmacology.

    PubMed

    Chhabria, Mahesh T; Brahmkshatriya, Pathik S; Mahajan, Bhushan M; Darji, Urvesh B; Shah, Gaurang B

    2012-07-01

    The present study describes ligand-based pharmacophore modeling of a series of structurally diverse acyl coenzyme A cholesterol acyltransferase inhibitors. Quantitative pharmacophore models were generated using HypoGen module of Discovery Studio 2.1, whereby the best pharmacophore model possessing two hydrophobic, one ring aromatic, and one hydrogen bond acceptor feature for inhibition of acyl coenzyme A cholesterol acyltransferase showed a very good correlation coefficient (r = 0.942) along with satisfactory cost analysis. Hypo1 was also validated by test set and cross-validation methods. Developed models were found to be predictive as indicated by low error values for test set molecules. Virtual screening against Maybridge database using Hypo1 was performed. The two most potent compounds (47 and 48; predicted IC₅₀ = 1 nM) of the retrieved hits were synthesized and biologically evaluated. These compounds showed 86% and 88% inhibition of acyl coenzyme A cholesterol acyltransferase (at 10 μg/mL) with IC₅₀ value of 3.6 and 2.5 nM, respectively. As evident from the close proximity of biological data to the predicted values, it can be concluded that the generated model (Hypo1) is a reliable and useful tool for lead optimization of novel acyl coenzyme A cholesterol acyltransferase inhibitors. © 2012 John Wiley & Sons A/S.

  11. Role of very-long-chain acyl-coenzyme A synthetase in X-linked adrenoleukodystrophy.

    PubMed

    Steinberg, S J; Kemp, S; Braiterman, L T; Watkins, P A

    1999-09-01

    X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by decreased ability of cells to activate (via very-long-chain acyl-coenzyme A synthetase [VLCS]) and subsequently degrade very-long-chain fatty acids in peroxisomes. It is noteworthy that the gene defective in X-ALD encodes ALDP, a peroxisomal membrane protein unrelated to VLCS. We cloned human VLCS (hVLCS) and found that peroxisomes from X-ALD fibroblasts contained immunoreactive hVLCS, refuting the earlier hypothesis that ALDP is required to anchor VLCS to the peroxisomal membrane. Furthermore, hVLCS was topographically oriented facing the peroxisomal matrix in both control and X-ALD fibroblasts, contradicting the alternative hypothesis that ALDP is required to translocate VLCS into peroxisomes. However, overexpression of both hVLCS and ALDP in X-ALD fibroblasts synergistically increased very-long-chain fatty acid beta-oxidation, indicating that these proteins interact functionally.

  12. Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.

    PubMed

    Mlícková, Katerina; Roux, Emeline; Athenstaedt, Karin; d'Andrea, Sabine; Daum, Günther; Chardot, Thierry; Nicaud, Jean-Marc

    2004-07-01

    Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.

  13. ACX3, a Novel Medium-Chain Acyl-Coenzyme A Oxidase from Arabidopsis

    PubMed Central

    Froman, Byron E.; Edwards, Patricia C.; Bursch, Adam G.; Dehesh, Katayoon

    2000-01-01

    In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene. PMID:10859203

  14. The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini

    PubMed Central

    Demirev, Atanas V.; Khanal, Anamika; Sedai, Bhishma R.; Lim, Si Kyu; Na, Min Kyun

    2010-01-01

    Streptomyces toxytricini produces lipstatin, a specific inhibitor of pancreatic lipase, which is derived from two fatty acid moieties with eight and 14 carbon atoms. The pccB gene locus in 10.6 kb fragment of S. toxytricini chromosomal DNA contains three genes for acyl-coenzyme A carboxylase (ACCase) complex accA3, pccB, and pccE that are presumed to be involved in secondary metabolism. The pccB gene encoding a β subunit of ACCase [carboxyltransferase (CT)] was identified upstream of pccE gene for a small protein of ε subunit. The accA3 encoding the α subunit of ACCase [biotin carboxylase (BC)] was also identified downstream of pccB gene. When the pccB and pccE genes were inactivated by homologous recombination, the lipstatin production was reduced as much as 80%. In contrast, the accumulation of another compound, tetradeca-5.8-dienoic acid (the major lipstatin precursor), was 4.5-fold increased in disruptant compared with wild-type. It implies that PccB of S. toxytricini is involved in the activation of octanoic acid to hexylmalonic acid for lipstatin biosynthesis. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2587-2) contains supplementary material, which is available to authorized users. PMID:20437235

  15. Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages.

    PubMed

    Khelef, N; Buton, X; Beatini, N; Wang, H; Meiner, V; Chang, T Y; Farese, R V; Maxfield, F R; Tabas, I

    1998-05-01

    Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.

  16. Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

    PubMed Central

    Ruprecht, Amanda; Maddox, Jaymie; Stirling, Alexander J.; Visaggio, Nicole

    2015-01-01

    ABSTRACT The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of Mycobacterium tuberculosis and Rhodococcus jostii RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the casC gene in R. jostii RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs. IMPORTANCE We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from M. tuberculosis and Rhodococcus jostii RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of

  17. Actinobacterial Acyl Coenzyme A Synthetases Involved in Steroid Side-Chain Catabolism

    PubMed Central

    Casabon, Israël; Swain, Kendra; Crowe, Adam M.

    2014-01-01

    Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 105 ± 0.03 × 105 M−1 s−1) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2′-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 105 ± 0.1 × 105 M−1 s−1 and 3.2 × 105 ± 0.3 × 105 M−1 s−1, respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and

  18. Kinetic characterization of the inhibition of acyl coenzyme A: steroid acyltransferases by tributyltin in the eastern mud snail (Ilyanassa obsoleta).

    PubMed

    Sternberg, Robin M; LeBlanc, Gerald A

    2006-06-30

    Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17beta-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (K(i)= approximately 9microM) and is a weaker, noncompetitive inhibitor of AEAT (K(i)= approximately 31microM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17beta-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17beta-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis-the fatty acid esterification of testosterone.

  19. Potential role of acyl-coenzyme A:cholesterol transferase (ACAT) Inhibitors as hypolipidemic and antiatherosclerosis drugs.

    PubMed

    Leon, Carlos; Hill, John S; Wasan, Kishor M

    2005-10-01

    Acyl-coenzyme A:cholesterol transferase (ACAT) is an integral membrane protein localized in the endoplasmic reticulum. ACAT catalyzes the formation of cholesteryl esters from cholesterol and fatty acyl coenzyme A. The cholesteryl esters are stored as cytoplasmic lipid droplets inside the cell. This process is very important to the organism as high cholesterol levels have been associated with cardiovascular disease. In mammals, two ACAT genes have been identified, ACAT1 and ACAT2. ACAT1 is ubiquitous and is responsible for cholesteryl ester formation in brain, adrenal glands, macrophages, and kidneys. ACAT2 is expressed in the liver and intestine. The inhibition of ACAT activity has been associated with decreased plasma cholesterol levels by suppressing cholesterol absorption and by diminishing the assembly and secretion of apolipoprotein B-containing lipoproteins such as very low density lipoprotein (VLDL). ACAT inhibition also prevents the conversion of macrophages into foam cells in the arterial walls, a critical event in the development of atherosclerosis. This review paper will focus on the role of ACAT in cholesterol metabolism, in particular as a target to develop novel therapeutic agents to control hypercholesterolemia, atherosclerosis, and Alzheimer's disease.

  20. The multiple acyl-coenzyme A dehydrogenation disorders, glutaric aciduria type II and ethylmalonic-adipic aciduria. Mitochondrial fatty acid oxidation, acyl-coenzyme A dehydrogenase, and electron transfer flavoprotein activities in fibroblasts.

    PubMed Central

    Amendt, B A; Rhead, W J

    1986-01-01

    The multiple acyl-coenzyme A (CoA) dehydrogenation disorders (MAD) include severe (S) and mild (M) variants, glutaric aciduria type II (MAD:S) and ethylmalonic-adipic aciduria (MAD:M). Intact MAD:M mitochondria oxidized [1-14C]octanoate, [1-14C]palmityl-CoA, and [1,5-14C]glutarate at 20-46% of control levels; MAD:S mitochondria oxidized these three substrates at 0.4-18% of control levels. In MAD:M mitochondria, acyl-CoA dehydrogenase (ADH) activities were similar to control, whereas MAD:S ADH activities ranged from 38% to 73% of control. Electron transfer flavoprotein (ETF) activities in five MAD:M cell lines ranged from 29 to 51% of control (P less than 0.01); ETF deficiency was the primary enzymatic defect in two MAD:M lines. In four MAD:S patients, ETF activities ranged from 3% to 6% of control (P less than 0.001); flavin adenine dinucleotide addition increased residual ETF activity from 4% to 21% of control in a single MAD:S line (P less than 0.01). Three MAD:S patients had ETF activities ranging from 33 to 53% of control; other investigators found deficient ETF-dehydrogenase activity in these MAD:S and three of our MAD:M cell lines. PMID:3722376

  1. Adrenoleukodystrophy protein enhances association of very long-chain acyl-coenzyme A synthetase with the peroxisome.

    PubMed

    Yamada, T; Taniwaki, T; Shinnoh, N; Uchiyama, A; Shimozawa, N; Ohyagi, Y; Asahara, H; Kira, J

    1999-02-01

    To clarify the function of adrenoleukodystrophy protein (ALDP) using our ALDP-deficient mice established by gene targeting. X-linked adrenoleukodystrophy (ALD) is characterized biochemically by the accumulation of very long-chain fatty acids (VLCFA) in tissues and body fluids, and is caused by impairment of peroxisomal beta-oxidation. In ALD, very long-chain acyl-coenzyme A synthetase (VLACS), which is necessary for peroxisomal beta-oxidation, does not function. The ALDP-deficient mice and C57BL/6J mice were used. VLACS or ALDP were transiently expressed by lipofection in murine fibroblasts, and VLCFA beta-oxidation was assayed. Liver peroxisomes were purified by sequential centrifugations and a Nycodenz gradient centrifugation. The peroxisomal localization of VLACS was compared between the mutant and control mice using a Western blot analysis. Impairment of VLCFA beta-oxidation in ALDP-deficient fibroblasts was not corrected by the additional expression of VLACS alone but was by the coexpression of VLACS and ALDP. Although the tissue-specific expression of VLACS was similar in ALDP-deficient and normal mice, peroxisomal VLACS was clearly lower in ALDP-deficient than in normal mice. ALDP plays a role in the peroxisomal localization of VLACS, and VLACS does not function unless localized in the peroxisome.

  2. Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.

    PubMed

    Waché, Y; Laroche, C; Bergmark, K; Møller-Andersen, C; Aguedo, M; Le Dall, M T; Wang, H; Nicaud, J M; Belin, J M

    2000-03-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140-5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.

  3. Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

    PubMed Central

    Waché, Yves; Laroche, Céline; Bergmark, Karin; Møller-Andersen, Charlotte; Aguedo, Mario; Le Dall, Marie-Thérèse; Wang, Huijie; Nicaud, Jean-Marc; Belin, Jean-Marc

    2000-01-01

    We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation. PMID:10698800

  4. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

    PubMed Central

    Fernández-Valverde, M; Reglero, A; Martinez-Blanco, H; Luengo, J M

    1993-01-01

    Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. Images PMID:8476289

  5. Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues.

    PubMed

    Sakashita, N; Miyazaki, A; Takeya, M; Horiuchi, S; Chang, C C; Chang, T Y; Takahashi, K

    2000-01-01

    To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.

  6. Anesthetic agents in patients with very long-chain acyl-coenzyme A dehydrogenase deficiency: a literature review.

    PubMed

    Redshaw, Charlotte; Stewart, Catherine

    2014-11-01

    Very long-chain acyl-coenzyme A dehydrongenase deficiency (VLCADD) is a rare disorder of fatty acid metabolism that renders sufferers susceptible to hypoglycemia, liver failure, cardiomyopathy, and rhabdomyolysis. The literature about the management of these patients is hugely conflicting, suggesting that both propofol and volatile anesthesia should be avoided. We have reviewed the literature and have concluded that the source papers do not support the statements that volatile anesthetic agents are unsafe. The reports on rhabdomyolysis secondary to anesthesia appear to be due to inadequate supply of carbohydrate not volatile agents. Catabolism must be avoided with minimal fasting, glucose infusions based on age and weight, and attenuation of emotional and physical stress. General anesthesia appears to be protective of stress-induced catabolism and may offer benefits in children and anxious patients over regional anesthesia. Propofol has not been demonstrated to be harmful in VLCADD but is presented in an emulsion containing very long-chain fatty acids which can cause organ lipidosis and itself can inhibit mitochondrial fatty acid metabolism. It is therefore not recommended. Suxamethonium-induced myalgia may mimic symptoms of rhabdomyolysis and cause raised CK therefore should be avoided. Opioids, NSAIDS, regional anesthesia, and local anesthetic techniques have all been used without complication.

  7. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis.

    PubMed

    Rowland, Owen; Zheng, Huanquan; Hepworth, Shelley R; Lam, Patricia; Jetter, Reinhard; Kunst, Ljerka

    2006-11-01

    A waxy cuticle that serves as a protective barrier against uncontrolled water loss and environmental damage coats the aerial surfaces of land plants. It is composed of a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very-long-chain fatty acids and their derivatives. We report here the molecular cloning and characterization of CER4, a wax biosynthetic gene from Arabidopsis (Arabidopsis thaliana). Arabidopsis cer4 mutants exhibit major decreases in stem primary alcohols and wax esters, and slightly elevated levels of aldehydes, alkanes, secondary alcohols, and ketones. This phenotype suggested that CER4 encoded an alcohol-forming fatty acyl-coenzyme A reductase (FAR). We identified eight FAR-like genes in Arabidopsis that are highly related to an alcohol-forming FAR expressed in seeds of jojoba (Simmondsia chinensis). Molecular characterization of CER4 alleles and genomic complementation revealed that one of these eight genes, At4g33790, encoded the FAR required for cuticular wax production. Expression of CER4 cDNA in yeast (Saccharomyces cerevisiae) resulted in the accumulation of C24:0 and C26:0 primary alcohols. Fully functional green fluorescent protein-tagged CER4 protein was localized to the endoplasmic reticulum in yeast cells by confocal microscopy. Analysis of gene expression by reverse transcription-PCR indicated that CER4 was expressed in leaves, stems, flowers, siliques, and roots. Expression of a beta-glucuronidase reporter gene driven by the CER4 promoter in transgenic plants was detected in epidermal cells of leaves and stems, consistent with a dedicated role for CER4 in cuticular wax biosynthesis. CER4 was also expressed in all cell types in the elongation zone of young roots. These data indicate that CER4 is an alcohol-forming FAR that has specificity for very-long-chain fatty acids and is responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in roots.

  8. Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rosen, Brandon C.; Dillon, Nicholas A.; Peterson, Nicholas D.; Minato, Yusuke

    2016-01-01

    ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis. PMID:27855077

  9. Structure of YciA from Haemophilus influenzae (HI0827), a Hexameric Broad Specificity Acyl-Coenzyme A Thioesterase

    SciTech Connect

    Willis, Mark A.; Zhuang, Zhihao; Song, Feng; Howard, Andrew; Dunaway-Mariano, Debra; Herzberg, Osnat

    2008-04-02

    The crystal structure of HI0827 from Haemophilus influenzae Rd KW20, initially annotated 'hypothetical protein' in sequence databases, exhibits an acyl-coenzyme A (acyl-CoA) thioesterase 'hot dog' fold with a trimer of dimers oligomeric association, a novel assembly for this enzyme family. In studies described in the preceding paper [Zhuang, Z., Song, F., Zhao, H., Li, L., Cao, J., Eisenstein, E., Herzberg, O., and Dunaway-Mariano, D. (2008) Biochemistry 47, 2789-2796], HI0827 is shown to be an acyl-CoA thioesterase that acts on a wide range of acyl-CoA compounds. Two substrate binding sites are located across the dimer interface. The binding sites are occupied by two CoA molecules, one with full occupancy and the second only partially occupied. The CoA molecules, acquired from HI0827-expressing Escherichia coli cells, remained tightly bound to the enzyme through the protein purification steps. The difference in CoA occupancies indicates a different substrate affinity for each of the binding sites, which in turn implies that the enzyme might be subject to allosteric regulation. Mutagenesis studies have shown that the replacement of the putative catalytic carboxylate Asp44 with an alanine residue abolishes activity. The impact of this mutation is seen in the crystal structure of D44A HI0827. Whereas the overall fold and assembly of the mutant protein are the same as those of the wild-type enzyme, the CoA ligands are absent. The dimer interface is perturbed, and the channel that accommodates the thioester acyl chain is more open and wider than that observed in the wild-type enzyme. A model of intact substrate bound to wild-type HI0827 provides a structural rationale for the broad substrate range.

  10. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    NASA Technical Reports Server (NTRS)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  11. Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

    NASA Technical Reports Server (NTRS)

    Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

  12. RP 64477: a potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase with low systemic bioavailability.

    PubMed

    Bello, A A; Bright, C; Burton, B J; Bush, R C; Casey, J H; Dron, D I; Facchini, V; Joannou, P P; Parrott, D P; Riddell, D; Roberts, S A; Williams, R J

    1996-02-23

    RP 64477 (N-butyl-3-(p-decyloxybenzamido)-4-(methylthio)benzamide) has been shown to be a potent inhibitor of the cholesterol esterifying enzyme Acyl-coenzyme A:cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) in intestinal, hepatic, adrenal, and arterial tissue preparations obtained from a range of animal species. Drug concentrations producing 50% inhibition of enzyme activity (IC50 values) ranged from 14-283 nM. Inhibition by RP 64477 in a rabbit intestinal enzyme preparation was shown to be non-competitive with respect to the substrate oleoyl-CoA. In whole cell assays using human intestinal (CaCo-2), hepatic HepG2) and monocytic (THP-1) cell lines, RP 64477 inhibited ACAT activity with IC50s of 113, 503, and 180 nM, respectively. RP 64477 (0.03% w/w by diet) reduced significantly cholesterol absorption in cholesterol/cholic acid-fed rats from 94+/- 8% to 65 +/- 4%. In cholesterol-fed rabbits, cholesterol absorption was reduced from 72 +/- 5% to 50 +/-5% and 44 +/- 5% at dose levels of 10 and 30 mg kg-1 b.i.d., respectively. Plasma cholesterol levels were reduced dose-dependently in both cholesterol/cholic-acid-fed rats and cholesterol-fed rabbits. Neither cholesterol absorption nor plasma cholesterol levels were reduced significantly in animals maintained on standard laboratory diets. Pharmacokinetic studies indicated that RP 64477 were very poorly absorbed following oral administration to rats. Plasma levels of drug were < 2 ng mL-1 following a dose of 2000 mg kg-1 p.o.. When radiolabelled RP 64477 was administered orally, limited absorption was indicated by the overwhelming elimination of radioactivity in the faces (96.4% of administered material) coupled with low renal clearance (0.6% of dose) and biliary excretion (0.05% of dose). In conclusion, this work shows that RP 64477 is a potent inhibitor of ACAT obtained from a range of animal species and man. Inhibition of cholesterol absorption and hypocholesterolaemic activity has been demonstrated in rats and

  13. Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

    PubMed Central

    Röttig, Annika; Strittmatter, Carl Simon; Schauer, Jennifer; Hiessl, Sebastian; Daniel, Rolf

    2016-01-01

    ABSTRACT Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli. The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCE A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected

  14. Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates†

    PubMed Central

    Fontes, Rui; Günther Sillero, Maria A.; Sillero, Antonio

    1998-01-01

    Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p4A) and adenosine 5′-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), and adenosine(5′)pentaphospho(5′)adenosine (Ap5A) are also substrates of the reaction yielding p4(d)A in the presence of tripolyphosphate (P3). UTP, CTP, and AMP are not substrates of the reaction. The Km values for ATP and P3 are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl2 or CoCl2 equimolecular with the sum of ATP and P3. The relative rates of synthesis of p4A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p4A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p4A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap4A (from ATP), Ap5A (from p4A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap4N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates). PMID:9620965

  15. Synthesis of fruity ethyl esters by acyl coenzyme A: alcohol acyltransferase and reverse esterase activities in Oenococcus oeni and Lactobacillus plantarum.

    PubMed

    Costello, P J; Siebert, T E; Solomon, M R; Bartowsky, E J

    2013-03-01

    To assess the abilities of commercial wine lactic acid bacteria (LAB) to synthesize potentially flavour active fatty acid ethyl esters and determine mechanisms involved in their production. Oenococcus oeni AWRI B551 produced significant levels of ethyl hexanoate and ethyl octanoate following growth in an ethanolic test medium, and ester formation generally increased with increasing pH (4.5 > 3.5), anaerobiosis and precursor supplementation. Cell-free extracts of commercial O. oeni strains and Lactobacillus plantarum AWRI B740 were also tested for ester-synthesizing capabilities in a phosphate buffer via: (i) acyl coenzyme A: alcohol acyltransferase (AcoAAAT) activity and (ii) reverse esterase activity. For both ester-synthesizing activities, strain-dependent variation was observed, with AcoAAAT activity generally greater than reverse esterase. Reverse esterase in O. oeni AWRI B551 also esterified 1-propanol to produce propyl octanoate, and deuterated substrates ([(2)H(6)]ethanol and [(2)H(15)]octanoic acid) to produce the fully deuterated ester, [(2)H(5)]ethyl [(2)H(15)]octanoate. Wine LAB exhibit ethyl ester-synthesizing capability and possess two different ester-synthesizing activities, one of which is associated with an acyl coenzyme A: alcohol acyltransferase. This study demonstrates that wine LAB exhibit enzyme activities that can augment the ethyl ester content of wine. This knowledge will facilitate greater control over the impacts of malolactic fermentation on the fruity sensory properties and quality of wine. © 2012 Australian Wine Research Institute © 2012 The Society for Applied Microbiology.

  16. Purification of a Jojoba Embryo Fatty Acyl-Coenzyme A Reductase and Expression of Its cDNA in High Erucic Acid Rapeseed

    PubMed Central

    Metz, James G.; Pollard, Michael R.; Anderson, Lana; Hayes, Thomas R.; Lassner, Michael W.

    2000-01-01

    The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes. PMID:10712526

  17. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

    PubMed Central

    Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

    2015-01-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876

  18. Shrinking the FadE Proteome of Mycobacterium tuberculosis: Insights into Cholesterol Metabolism through Identification of an α2β2 Heterotetrameric Acyl Coenzyme A Dehydrogenase Family

    PubMed Central

    Wipperman, Matthew F.; Yang, Meng; Thomas, Suzanne T.

    2013-01-01

    The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism. PMID:23836861

  19. Shrinking the FadE proteome of Mycobacterium tuberculosis: insights into cholesterol metabolism through identification of an α2β2 heterotetrameric acyl coenzyme A dehydrogenase family.

    PubMed

    Wipperman, Matthew F; Yang, Meng; Thomas, Suzanne T; Sampson, Nicole S

    2013-10-01

    The ability of the pathogen Mycobacterium tuberculosis to metabolize steroids like cholesterol and the roles that these compounds play in the virulence and pathogenesis of this organism are increasingly evident. Here, we demonstrate through experiments and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl coenzyme A (acyl-CoA) dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. FadE26-FadE27 catalyzes the dehydrogenation of 3β-hydroxy-chol-5-en-24-oyl-CoA, an analog of the 5-carbon side chain cholesterol degradation intermediate. Genes encoding the α2β2 heterotetrameric ACAD structures are present in multiple regions of the M. tuberculosis genome, and subsets of these genes are regulated by four different transcriptional repressors or activators: KstR1 (also known as KstR), KstR2, Mce3R, and SigE. Homologous ACAD gene pairs are found in other Actinobacteria, as well as Proteobacteria. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four subpathways of cholesterol metabolism.

  20. Structural requirements for charged lipid molecules to directly increase or suppress K+ channel activity in smooth muscle cells. Effects of fatty acids, lysophosphatidate, acyl coenzyme A and sphingosine

    PubMed Central

    1994-01-01

    We determined the structural features necessary for fatty acids to exert their action on K+ channels of gastric smooth muscle cells. Examination of the effects of a variety of synthetic and naturally occurring lipid compounds on K+ channel activity in cell-attached and excised membrane patches revealed that negatively charged analogs of medium to long chain fatty acids (but not short chain analogs) as well as certain other negatively charged lipids activate the channels. In contrast, positively charged, medium to long chain analogs suppress activity, and neutral analogs are without effect. The key requirements for effective compounds seem to be a sufficiently hydrophobic domain and the presence of a charged group. Furthermore, those negatively charged compounds unable to "flip" across the bilayer are effective only when applied at the cytosolic surface of the membrane, suggesting that the site of fatty acid action is also located there. Finally, because some of the effective compounds, for example, the fatty acids themselves, lysophosphatidate, acyl Coenzyme A, and sphingosine, are naturally occurring substances and can be liberated by agonist- activated or metabolic enzymes, they may act as second messengers targeting ion channels. PMID:8195783

  1. Mutation analysis of very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency: Identification and characterization of mutant VLCAD cDNAs from four patients

    SciTech Connect

    Souri, Masayoshi; Aoyama, Toshifumi; Hashimoto, Takashi

    1996-01-01

    Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is a newly identified disease. A 105-bp deletion in the VLCAD cDNA in two patients has been reported, and detailed molecular characterization of this disease has remained to be done. We report here five mutations identified in four patients: a 135-bp deletion encompassing bases 343-477, a C-1837-to-T transition (R613W), 3-bp deletions at the nucleotide positions 388-390 (E130del) and 895-897 (K299del), and an A-1144-to-C transversion (K382Q). Sequencing of genomic DNA amplified by PCR revealed a 135-bp deletion caused by exon skipping due to a 1-bp deletion in a 3{prime} splice site of an intron. In cDNA expression experiments using Chinese hamster ovary (CHO) cells, we found that each of the mRNAs derived from E130del and K299del clones were unstable and that translation products from R613W, E130del, K299del, and K382Q clones were labile. Each of R613W, E130del, K299del, and K382Q proteins expressed in CHO cells appeared abnormal in dimer assembly, as shown in gel-filtration analysis. VLCAD activity was not detected in mutants` transfectants. Thus, we verified that all five mutations identified in these four patients were disease-causing alterations. 34 refs., 7 figs., 4 tabs.

  2. Importance of acyl-coenzyme A:cholesterol acyltransferase 1/2 dual inhibition for anti-atherosclerotic potency of pactimibe.

    PubMed

    Kitayama, Ken; Tanimoto, Tatsuo; Koga, Teiichiro; Terasaka, Naoki; Fujioka, Tomoyuki; Inaba, Toshimori

    2006-07-01

    Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC50s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (Ki value: 5.6 microM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC50: 6.7 microM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.

  3. Two Activities of Long-Chain Acyl-Coenzyme A Synthetase Are Involved in Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis1

    PubMed Central

    Jessen, Dirk; Roth, Charlotte; Wiermer, Marcel

    2015-01-01

    In plants, fatty acids are synthesized within the plastid and need to be distributed to the different sites of lipid biosynthesis within the cell. Free fatty acids released from the plastid need to be converted to their corresponding coenzyme A thioesters to become metabolically available. This activation is mediated by long-chain acyl-coenzyme A synthetases (LACSs), which are encoded by a family of nine genes in Arabidopsis (Arabidopsis thaliana). So far, it has remained unclear which of the individual LACS activities are involved in making plastid-derived fatty acids available to cytoplasmic glycerolipid biosynthesis. Because of its unique localization at the outer envelope of plastids, LACS9 was regarded as a candidate for linking plastidial fatty export and cytoplasmic use. However, data presented in this study show that LACS9 is involved in fatty acid import into the plastid. The analyses of mutant lines revealed strongly overlapping functions of LACS4 and LACS9 in lipid trafficking from the endoplasmic reticulum to the plastid. In vivo labeling experiments with lacs4 lacs9 double mutants suggest strongly reduced synthesis of endoplasmic reticulum-derived lipid precursors, which are required for the biosynthesis of glycolipids in the plastids. In conjunction with this defect, double-mutant plants accumulate significant amounts of linoleic acid in leaf tissue. PMID:25540329

  4. Regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) synthesis, degradation, and translocation by high-density lipoprotein(2) at a low concentration.

    PubMed

    Li, L; Pownall, H J

    2000-12-01

    (,Although plasma HDL(2) cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL(2) at a low concentration (< or = 40 microg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL(2) on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL(2) at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL(2), however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL(2) promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL(2) is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.

  5. The Endoplasmic Reticulum-Associated Maize GL8 Protein Is a Component of the Acyl-Coenzyme A Elongase Involved in the Production of Cuticular Waxes1

    PubMed Central

    Xu, Xiaojie; Dietrich, Charles R.; Lessire, Rene; Nikolau, Basil J.; Schnable, Patrick S.

    2002-01-01

    The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a β-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes. PMID:11891248

  6. Acyl-coenzyme A oxidases 1 and 3 in brown trout (Salmo trutta f. fario): Can peroxisomal fatty acid β-oxidation be regulated by estrogen signaling?

    PubMed

    Madureira, Tânia Vieira; Castro, L Filipe C; Rocha, Eduardo

    2016-02-01

    Acyl-coenzyme A oxidases 1 (Acox1) and 3 (Acox3) are key enzymes in the regulation of lipid homeostasis. Endogenous and exogenous factors can disrupt their normal expression/activity. This study presents for the first time the isolation and characterization of Acox1 and Acox3 in brown trout (Salmo trutta f. fario). Additionally, as previous data point to the existence of a cross-talk between two nuclear receptors, namely peroxisome proliferator-activated receptors and estrogen receptors, it was here evaluated after in vitro exposures of trout hepatocytes the interference caused by ethynylestradiol in the mRNA levels of an inducible (by peroxisome proliferators) and a non-inducible oxidase. The isolated Acox1 and Acox3 show high levels of sequence conservation compared to those of other teleosts. Additionally, it was found that Acox1 has two alternative splicing isoforms, corresponding to 3I and 3II isoforms of exon 3 splicing variants. Both isoforms display tissue specificity, with Acox1-3II presenting a more ubiquitous expression in comparison with Acox1-3I. Acox3 was expressed in almost all brown trout tissues. According to real-time PCR data, the highest estrogenic stimulus was able to cause a down-regulation of Acox1 and an up-regulation of Acox3. So, for Acox1 we found a negative association between an estrogenic input and a directly activated PPARα target gene. In conclusion, changes in hormonal estrogenic stimulus may impact the mobilization of hepatic lipids to the gonads, with ultimate consequences in reproduction. Further studies using in vivo assays will be fundamental to clarify these issues.

  7. Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A.

    PubMed

    Dunn, Michael F; Araíza, Gisela; Mora, Jaime

    2004-02-01

    Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K(m) and V(max) values of 0.064 mM and 2885 nmol min(-1) (mg protein)(-1), respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K(m) values of 0.392 and 0.144 mM, respectively, and V(max) values of 423 and 268 nmol min(-1) (mg protein)(-1), respectively. K(+), or Cs(+) markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing alpha subunit and a 45 kDa biotin-free beta subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.

  8. FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.)

    PubMed Central

    Wang, Yong; Wang, Meiling; Sun, Yulin; Wang, Yanting; Li, Tingting; Chai, Guaiqiang; Jiang, Wenhui; Shan, Liwei; Li, Chunlian; Xiao, Enshi; Wang, Zhonghua

    2015-01-01

    A waxy cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage coats the aerial surfaces of land plants. It comprises a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. Results show that primary alcohols are the major components of bread wheat (Triticum aestivum L.) leaf blade cuticular waxes. Here, the characterization of TaFAR5 from wheat cv Xinong 2718, which is allelic to TAA1b, an anther-specific gene, is reported. Evidence is presented for a new function for TaFAR5 in the biosynthesis of primary alcohols of leaf blade cuticular wax in wheat. Expression of TaFAR5 cDNA in yeast (Saccharomyces cerevisiae) led to production of C22:0 primary alcohol. The transgenic expression of TaFAR5 in tomato (Solanum lycopersicum) cv MicroTom leaves resulted in the accumulation of C26:0, C28:0, and C30:0 primary alcohols. TaFAR5 encodes an alcohol-forming fatty acyl-coenzyme A reductase (FAR). Expression analysis revealed that TaFAR5 was expressed at high levels in the leaf blades, anthers, pistils, and seeds. Fully functional green fluorescent protein-tagged TaFAR5 protein was localized to the endoplasmic reticulum (ER), the site of primary alcohol biosynthesis. SDS–PAGE analysis indicated that the TaFAR5 protein possessed a molecular mass of 58.4kDa, and it was also shown that TaFAR5 transcript levels were regulated in response to drought, cold, and abscisic acid (ABA). Overall, these data suggest that TaFAR5 plays an important role in the synthesis of primary alcohols in wheat leaf blade. PMID:25468933

  9. FAR5, a fatty acyl-coenzyme A reductase, is involved in primary alcohol biosynthesis of the leaf blade cuticular wax in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yong; Wang, Meiling; Sun, Yulin; Wang, Yanting; Li, Tingting; Chai, Guaiqiang; Jiang, Wenhui; Shan, Liwei; Li, Chunlian; Xiao, Enshi; Wang, Zhonghua

    2015-03-01

    A waxy cuticle that serves as a protective barrier against non-stomatal water loss and environmental damage coats the aerial surfaces of land plants. It comprises a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. Results show that primary alcohols are the major components of bread wheat (Triticum aestivum L.) leaf blade cuticular waxes. Here, the characterization of TaFAR5 from wheat cv Xinong 2718, which is allelic to TAA1b, an anther-specific gene, is reported. Evidence is presented for a new function for TaFAR5 in the biosynthesis of primary alcohols of leaf blade cuticular wax in wheat. Expression of TaFAR5 cDNA in yeast (Saccharomyces cerevisiae) led to production of C22:0 primary alcohol. The transgenic expression of TaFAR5 in tomato (Solanum lycopersicum) cv MicroTom leaves resulted in the accumulation of C26:0, C28:0, and C30:0 primary alcohols. TaFAR5 encodes an alcohol-forming fatty acyl-coenzyme A reductase (FAR). Expression analysis revealed that TaFAR5 was expressed at high levels in the leaf blades, anthers, pistils, and seeds. Fully functional green fluorescent protein-tagged TaFAR5 protein was localized to the endoplasmic reticulum (ER), the site of primary alcohol biosynthesis. SDS-PAGE analysis indicated that the TaFAR5 protein possessed a molecular mass of 58.4kDa, and it was also shown that TaFAR5 transcript levels were regulated in response to drought, cold, and abscisic acid (ABA). Overall, these data suggest that TaFAR5 plays an important role in the synthesis of primary alcohols in wheat leaf blade.

  10. The effect of inhibition of acyl coenzyme A-cholesterol acyltransferase (ACAT) on exercise performance in patients with peripheral arterial disease.

    PubMed

    Hiatt, William R; Klepack, Ellen; Nehler, Mark; Regensteiner, Judith G; Blue, John; Imus, James; Criqui, Michael H

    2004-11-01

    This study tested the hypothesis that avasimibe, an inhibitor of acyl coenzyme A-cholesterol acyltransferase (ACAT), would improve treadmill exercise performance in patients with claudication secondary to peripheral arterial disease (PAD). Four hundred and forty-two patients with PAD (ankle-brachial index in the index leg of < or =0.90 with a > or =20% reduction post-exercise) were enrolled from 39 centers in the USA. Patients were randomized to receive oral avasimibe 50 mg, 250 mg, 750 mg or placebo for a treatment period of 12 months. Changes from baseline in peak walking time (PWT) using a graded treadmill protocol were compared among groups after 6 and 12 months of treatment. Individual group comparisons were considered statistically significant if p < 0.0245 for the 50 mg and 250 mg groups and p < 0.001 for the 750 mg group. Patients randomized to the 50 mg group experienced a 0.76 min net increase over placebo in PWT, but this did not reach the pre-specified level of statistical significance (Hochberg procedure p = 0.027) using ANCOVA after 12 months of treatment after adjusting for multiple comparisons. This trend in PWT was supported by the changes in treadmill initial claudication time (ICT) (p = 0.026) and Walking Impairment Questionnaire (WIQ) walking distance score (p = 0.058). The 250 mg and 750 mg avasimibe dose groups failed to demonstrate an improvement in PWT over placebo after 6 months of treatment. In conclusion, while the ACAT inhibitor avasimibe did not show clear evidence of benefit on treadmill exercise performance in patients with PAD, the results add to our knowledge of the impact of treatments directed at atherosclerosis on functional endpoints.

  11. CER4 Encodes an Alcohol-Forming Fatty Acyl-Coenzyme A Reductase Involved in Cuticular Wax Production in Arabidopsis1[W

    PubMed Central

    Rowland, Owen; Zheng, Huanquan; Hepworth, Shelley R.; Lam, Patricia; Jetter, Reinhard; Kunst, Ljerka

    2006-01-01

    A waxy cuticle that serves as a protective barrier against uncontrolled water loss and environmental damage coats the aerial surfaces of land plants. It is composed of a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very-long-chain fatty acids and their derivatives. We report here the molecular cloning and characterization of CER4, a wax biosynthetic gene from Arabidopsis (Arabidopsis thaliana). Arabidopsis cer4 mutants exhibit major decreases in stem primary alcohols and wax esters, and slightly elevated levels of aldehydes, alkanes, secondary alcohols, and ketones. This phenotype suggested that CER4 encoded an alcohol-forming fatty acyl-coenzyme A reductase (FAR). We identified eight FAR-like genes in Arabidopsis that are highly related to an alcohol-forming FAR expressed in seeds of jojoba (Simmondsia chinensis). Molecular characterization of CER4 alleles and genomic complementation revealed that one of these eight genes, At4g33790, encoded the FAR required for cuticular wax production. Expression of CER4 cDNA in yeast (Saccharomyces cerevisiae) resulted in the accumulation of C24:0 and C26:0 primary alcohols. Fully functional green fluorescent protein-tagged CER4 protein was localized to the endoplasmic reticulum in yeast cells by confocal microscopy. Analysis of gene expression by reverse transcription-PCR indicated that CER4 was expressed in leaves, stems, flowers, siliques, and roots. Expression of a β-glucuronidase reporter gene driven by the CER4 promoter in transgenic plants was detected in epidermal cells of leaves and stems, consistent with a dedicated role for CER4 in cuticular wax biosynthesis. CER4 was also expressed in all cell types in the elongation zone of young roots. These data indicate that CER4 is an alcohol-forming FAR that has specificity for very-long-chain fatty acids and is responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in roots. PMID:16980563

  12. Genetic basis for correction of very-long-chain acyl-coenzyme A dehydrogenase deficiency by bezafibrate in patient fibroblasts: toward a genotype-based therapy.

    PubMed

    Gobin-Limballe, S; Djouadi, F; Aubey, F; Olpin, S; Andresen, B S; Yamaguchi, S; Mandel, H; Fukao, T; Ruiter, J P N; Wanders, R J A; McAndrew, R; Kim, J J; Bastin, J

    2007-12-01

    Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an inborn mitochondrial fatty-acid beta-oxidation (FAO) defect associated with a broad mutational spectrum, with phenotypes ranging from fatal cardiopathy in infancy to adolescent-onset myopathy, and for which there is no established treatment. Recent data suggest that bezafibrate could improve the FAO capacities in beta-oxidation-deficient cells, by enhancing the residual level of mutant enzyme activity via gene-expression stimulation. Since VLCAD-deficient patients frequently harbor missense mutations with unpredictable effects on enzyme activity, we investigated the response to bezafibrate as a function of genotype in 33 VLCAD-deficient fibroblasts representing 45 different mutations. Treatment with bezafibrate (400 microM for 48 h) resulted in a marked increase in FAO capacities, often leading to restoration of normal values, for 21 genotypes that mainly corresponded to patients with the myopathic phenotype. In contrast, bezafibrate induced no changes in FAO for 11 genotypes corresponding to severe neonatal or infantile phenotypes. This pattern of response was not due to differential inductions of VLCAD messenger RNA, as shown by quantitative real-time polymerase chain reaction, but reflected variable increases in measured VLCAD residual enzyme activity in response to bezafibrate. Genotype cross-analysis allowed the identification of alleles carrying missense mutations, which could account for these different pharmacological profiles and, on this basis, led to the characterization of 9 mild and 11 severe missense mutations. Altogether, the responses to bezafibrate reflected the severity of the metabolic blockage in various genotypes, which appeared to be correlated with the phenotype, thus providing a new approach for analysis of genetic heterogeneity. Finally, this study emphasizes the potential of bezafibrate, a widely prescribed hypolipidemic drug, for the correction of VLCAD deficiency and

  13. FadD from Pseudomonas putida CA-3 Is a True Long-Chain Fatty Acyl Coenzyme A Synthetase That Activates Phenylalkanoic and Alkanoic Acids▿ †

    PubMed Central

    Hume, Aisling R.; Nikodinovic-Runic, Jasmina; O'Connor, Kevin E.

    2009-01-01

    A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher kcat and Km values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3ΔfadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3ΔfadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3ΔfadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longer-monomer units into the PHA polymer. PMID:19820085

  14. The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR

    PubMed Central

    van Aalten, Daan M.F.; DiRusso, Concetta C.; Knudsen, Jens

    2001-01-01

    FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli. The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-CoA. The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule >30 Å away from the DNA binding site affects transcriptional regulation. Here, we describe the structures of the FadR–operator and FadR– myristoyl-CoA binary complexes. The FadR–DNA complex reveals a novel winged helix–turn–helix protein–DNA interaction, involving sequence-specific contacts from the wing to the minor groove. Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 Å. The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 Å in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation. PMID:11296236

  15. 221 Newborn-Screened Neonates with Medium-Chain Acyl-Coenzyme A Dehydrogenase Deficiency: Findings from the Inborn Errors of Metabolism Collaborative

    PubMed Central

    Bentler, Kristi; Zhai, Shaohui; Elsbecker, Sara A.; Arnold, Georgianne L.; Burton, Barbara K.; Vockley, Jerry; Cameron, Cynthia A.; Hiner, Sally J.; Edick, Mathew J.; Berry, Susan A.

    2016-01-01

    INTRODUCTION There is limited understanding of relationships between genotype, phenotype and other conditions contributing to health in neonates with medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD) identified through newborn screening. METHODS Retrospective analysis of comprehensive data from a cohort of 221 newborn-screened subjects identified as affected with MCADD in the Inborn Errors of Metabolism – Information System (IBEM-IS), a long term follow-up database of the Inborn Errors of Metabolism Collaborative, was performed. RESULTS The average age at notification of first newborn screen results to primary care or metabolic providers was 7.45 days. The average octanoylcarnitine (C8) value on first newborn screen was 11.2 umol/L (median 8.6, range 0.36–43.91). A higher C8 level correlated with an earlier first subspecialty visit. Subjects with low birth weight had significantly lower C8 values. Significantly higher C8 values were found in symptomatic newborns, in newborns with abnormal lab testing in addition to newborn screening and/or diagnostic tests, and in subjects homozygous for the c.985A>G ACADM gene mutation or compound heterozygous for the c.985A>G mutation and deletions or other known highly deleterious mutations. Subjects with neonatal symptoms, or neonatal abnormal labs, or neonatal triggers were more likely to have at least one copy of the severe c.985A>G ACADM gene mutation. C8 and genotype category were significant predictors of the likelihood of having neonatal symptoms. Neonates with select triggers were more likely to have symptoms and laboratory abnormalities. CONCLUSIONS This collaborative study is the first in the United States to describe health associations of a large cohort of newborn-screened neonates identified as affected with MCADD. The IBEM-IS has utility as a platform to better understand the characteristics of individuals with newborn-screened conditions and their follow-up interactions with the health system. PMID

  16. Mangiferin treatment inhibits hepatic expression of acyl-coenzyme A:diacylglycerol acyltransferase-2 in fructose-fed spontaneously hypertensive rats: a link to amelioration of fatty liver

    SciTech Connect

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang; Chonan, Ritsu; Yamahara, Johji; Wang, Jianwei; Li, Yuhao

    2014-10-15

    Mangiferin, a xanthone glucoside, and its associated traditional herbs have been demonstrated to improve abnormalities of lipid metabolism. However, its underlying mechanisms remain largely unclear. This study investigated the anti-steatotic effect of mangiferin in fructose-fed spontaneously hypertensive rat (SHR)s that have a mutation in sterol regulatory element binding protein (SREBP)-1. The results showed that co-administration of mangiferin (15 mg/kg, once daily, by oral gavage) over 7 weeks dramatically diminished fructose-induced increases in hepatic triglyceride content and Oil Red O-stained area in SHRs. However, blood pressure, fructose and chow intakes, white adipose tissue weight and metabolic parameters (plasma concentrations of glucose, insulin, triglyceride, total cholesterol and non-esterified fatty acids) were unaffected by mangiferin treatment. Mechanistically, mangiferin treatment suppressed acyl-coenzyme A:diacylglycerol acyltransferase (DGAT)-2 expression at the mRNA and protein levels in the liver. In contrast, mangiferin treatment was without effect on hepatic mRNA and/or protein expression of SREBP-1/1c, carbohydrate response element binding protein, liver pyruvate kinase, fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase-1, DGAT-1, monoacyglycerol acyltransferase-2, microsomal triglyceride transfer protein, peroxisome proliferator-activated receptor-alpha, carnitine palmitoyltransferase-1 and acyl-CoA oxidase. Collectively, our results suggest that mangiferin treatment ameliorates fatty liver in fructose-fed SHRs by inhibiting hepatic DGAT-2 that catalyzes the final step in triglyceride biosynthesis. The anti-steatotic effect of mangiferin may occur independently of the hepatic signals associated with de novo fatty acid synthesis and oxidation. - Highlights: • We investigated the anti-steatotic effect of mangiferin (MA) in fructose-fed SHR. • MA (15 mg/kg/day for 7 weeks) ameliorated fructose-induced fatty liver in

  17. Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

    PubMed Central

    Uthoff, Stefan; Stöveken, Tim; Weber, Nikolaus; Vosmann, Klaus; Klein, Erika; Kalscheuer, Rainer; Steinbüchel, Alexander

    2005-01-01

    The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5′His6WS/DGAT comprising an N-terminal His6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5′His6atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein−1 min−1 was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5′His6WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1ΩKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry

  18. 221 newborn-screened neonates with medium-chain acyl-coenzyme A dehydrogenase deficiency: Findings from the Inborn Errors of Metabolism Collaborative.

    PubMed

    Bentler, Kristi; Zhai, Shaohui; Elsbecker, Sara A; Arnold, Georgianne L; Burton, Barbara K; Vockley, Jerry; Cameron, Cynthia A; Hiner, Sally J; Edick, Mathew J; Berry, Susan A

    2016-09-01

    There is limited understanding of relationships between genotype, phenotype and other conditions contributing to health in neonates with medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD) identified through newborn screening. Retrospective analysis of comprehensive data from a cohort of 221 newborn-screened subjects identified as affected with MCADD in the Inborn Errors of Metabolism - Information System (IBEM-IS), a long term follow-up database of the Inborn Errors of Metabolism Collaborative, was performed. The average age at notification of first newborn screen results to primary care or metabolic providers was 7.45days. The average octanoylcarnitine (C8) value on first newborn screen was 11.2μmol/L (median 8.6, range 0.36-43.91). A higher C8 level correlated with an earlier first subspecialty visit. Subjects with low birth weight had significantly lower C8 values. Significantly higher C8 values were found in symptomatic newborns, in newborns with abnormal lab testing in addition to newborn screening and/or diagnostic tests, and in subjects homozygous for the c.985A>G ACADM gene mutation or compound heterozygous for the c.985A>G mutation and deletions or other known highly deleterious mutations. Subjects with neonatal symptoms, or neonatal abnormal labs, or neonatal triggers were more likely to have at least one copy of the severe c.985A>G ACADM gene mutation. C8 and genotype category were significant predictors of the likelihood of having neonatal symptoms. Neonates with select triggers were more likely to have symptoms and laboratory abnormalities. This collaborative study is the first in the United States to describe health associations of a large cohort of newborn-screened neonates identified as affected with MCADD. The IBEM-IS has utility as a platform to better understand the characteristics of individuals with newborn-screened conditions and their follow-up interactions with the health system. Copyright © 2016 Elsevier Inc. All rights

  19. Three Arabidopsis Fatty Acyl-Coenzyme A Reductases, FAR1, FAR4, and FAR5, Generate Primary Fatty Alcohols Associated with Suberin Deposition1[C][W][OA

    PubMed Central

    Domergue, Frédéric; Vishwanath, Sollapura J.; Joubès, Jérôme; Ono, Jasmine; Lee, Jennifer A.; Bourdon, Matthieu; Alhattab, Reem; Lowe, Christine; Pascal, Stéphanie; Lessire, René; Rowland, Owen

    2010-01-01

    Suberin is a protective hydrophobic barrier consisting of phenolics, glycerol, and a variety of fatty acid derivatives, including C18:0-C22:0 primary fatty alcohols. An eight-member gene family encoding alcohol-forming fatty acyl-coenzyme A reductases (FARs) has been identified in Arabidopsis (Arabidopsis thaliana). Promoter-driven expression of the β-glucuronidase reporter gene indicated that three of these genes, FAR1(At5g22500), FAR4(At3g44540), and FAR5(At3g44550), are expressed in root endodermal cells. The three genes were transcriptionally induced by wounding and salt stress. These patterns of gene expression coincide with known sites of suberin deposition. We then characterized a set of mutants with T-DNA insertions in FAR1, FAR4, or FAR5 and found that the suberin compositions of roots and seed coats were modified in each far mutant. Specifically, C18:0-OH was reduced in far5-1, C20:0-OH was reduced in far4-1, and C22:0-OH was reduced in far1-1. We also analyzed the composition of polymer-bound lipids of leaves before and after wounding and found that the basal levels of C18:0-C22:0 primary alcohols in wild-type leaves were increased by wounding. In contrast, C18:0-OH and C22:0-OH were not increased by wounding in far5-1 and far1-1 mutants, respectively. Heterologous expression of FAR1, FAR4, and FAR5 in yeast confirmed that they are indeed active alcohol-forming FARs with distinct, but overlapping, chain length specificities ranging from C18:0 to C24:0. Altogether, these results indicate that Arabidopsis FAR1, FAR4, and FAR5 generate the fatty alcohols found in root, seed coat, and wound-induced leaf tissue. PMID:20571114

  20. Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring.

    PubMed

    Blachnio-Zabielska, Agnieszka U; Koutsari, Christina; Jensen, Michael D

    2011-08-15

    Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13

  1. Transcriptional Regulation by the Short-Chain Fatty Acyl Coenzyme A Regulator (ScfR) PccR Controls Propionyl Coenzyme A Assimilation by Rhodobacter sphaeroides

    PubMed Central

    Carter, Michael S.

    2015-01-01

    ABSTRACT Propionyl coenzyme A (propionyl-CoA) assimilation by Rhodobacter sphaeroides proceeds via the methylmalonyl-CoA pathway. The activity of the key enzyme of the pathway, propionyl-CoA carboxylase (PCC), was upregulated 20-fold during growth with propionate compared to growth with succinate. Because propionyl-CoA is an intermediate in acetyl-CoA assimilation via the ethylmalonyl-CoA pathway, acetate growth also requires the methylmalonyl-CoA pathway. PCC activities were upregulated 8-fold in extracts of acetate-grown cells compared to extracts of succinate-grown cells. The upregulation of PCC activities during growth with propionate or acetate corresponded to increased expression of the pccB gene, which encodes a subunit of PCC. PccR (RSP_2186) was identified to be a transcriptional regulator required for the upregulation of pccB transcript levels and, consequently, PCC activity: growth substrate-dependent regulation was lost when pccR was inactivated by an in-frame deletion. In the pccR mutant, lacZ expression from a 215-bp plasmid-borne pccB upstream fragment including 27 bp of the pccB coding region was also deregulated. A loss of regulation as a result of mutations in the conserved motifs TTTGCAAA-X4-TTTGCAAA in the presence of PccR allowed the prediction of a possible operator site. PccR, together with homologs from other organisms, formed a distinct clade within the family of short-chain fatty acyl coenzyme A regulators (ScfRs) defined here. Some members from other clades within the ScfR family have previously been shown to be involved in regulating acetyl-CoA assimilation by the glyoxylate bypass (RamB) or propionyl-CoA assimilation by the methylcitrate cycle (MccR). IMPORTANCE Short-chain acyl-CoAs are intermediates in essential biosynthetic and degradative pathways. The regulation of their accumulation is crucial for appropriate cellular function. This work identifies a regulator (PccR) that prevents the accumulation of propionyl-CoA by controlling

  2. Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

    PubMed Central

    Tobin, M B; Fleming, M D; Skatrud, P L; Miller, J R

    1990-01-01

    The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa. Images PMID:2120195

  3. Immunodepletion experiments suggest that acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines.

    PubMed

    Lee, O; Chang, C C; Lee, W; Chang, T Y

    1998-08-01

    The first acyl-coenzyme A:cholesterol acyltransferase (ACAT) cDNA cloned and expressed in 1993 is designated as ACAT-1. In various human tissue homogenates, ACAT-1 protein is effectively solubilized with retention of enzymatic activity by the detergent CHAPS along with high salt. After using anti-ACAT-1 antibodies to quantitatively remove ACAT-1 protein from the solubilized enzyme, measuring the residual ACAT activity remaining in the immunodepleted supernatants allows us to assess the functional significance of ACAT-1 protein in various human tissues. The results showed that ACAT activity was immunodepleted 90% in liver (83% in hepatocytes), 98% in adrenal gland, 91% in macrophages, 80% in kidney, and 19% in intestines, suggesting that ACAT-1 protein plays a major catalytic role in all of the human tissue/cell homogenates examined except intestines. Intestinal ACAT activity is largely resistant to immunodepletion and is much more sensitive to inhibition by the ACAT inhibitor Dup 128 than liver ACAT activity.

  4. Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

    PubMed Central

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. PMID:24163338

  5. Characterization of two members among the five ADP-forming acyl coenzyme A (Acyl-CoA) synthetases reveals the presence of a 2-(Imidazol-4-yl)acetyl-CoA synthetase in Thermococcus kodakarensis.

    PubMed

    Awano, Tomotsugu; Wilming, Anja; Tomita, Hiroya; Yokooji, Yuusuke; Fukui, Toshiaki; Imanaka, Tadayuki; Atomi, Haruyuki

    2014-01-01

    The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.

  6. Bioconversion of α-linolenic acid to n-3 LCPUFA and expression of PPAR-alpha, acyl Coenzyme A oxidase 1 and carnitine acyl transferase I are incremented after feeding rats with α-linolenic acid-rich oils.

    PubMed

    González-Mañán, Daniel; Tapia, Gladys; Gormaz, Juan Guillermo; D'Espessailles, Amanda; Espinosa, Alejandra; Masson, Lilia; Varela, Patricia; Valenzuela, Alfonso; Valenzuela, Rodrigo

    2012-07-01

    High dietary intake of n-6 fatty acids in relation to n-3 fatty acids may generate health disorders, such as cardiovascular and other chronic diseases. Fish consumption rich in n-3 fatty acids is low in Latin America, it being necessary to seek other alternatives to provide α-linolenic acid (ALA), precursor of n-3 LCPUFA (EPA and DHA). Two innovative oils were assayed, chia (Salvia hispanica) and rosa mosqueta (Rosa rubiginosa). This study evaluated hepatic bioconversion of ALA to EPA and DHA, expression of PPAR-α, acyl-Coenzyme A oxidase 1 (ACOX1) and carnitine acyltransferase I (CAT-I), and accumulation of EPA and DHA in plasma and adipose tissue in Sprague-Dawley rats. Three experimental groups were fed 21 days: sunflower oil (SFO, control); chia oil (CO); rosa mosqueta oil (RMO). Fatty acid composition of total lipids and phospholipids from plasma, hepatic and adipose tissue was assessed by gas-liquid chromatography and TLC. Expression of PPAR-α (RT-PCR) and ACOX1 and CAT-I (Western blot). CO and RMO increased plasma, hepatic and adipose tissue levels of ALA, EPA and DHA and decreased n-6:n-3 ratio compared to SFO (p < 0.05, One-way ANOVA and Newman-Keuls test). CO increased levels of ALA and EPA compared to RMO (p < 0.05). No significant differences were observed for DHA levels. CO also increased the expression of PPAR-α, ACOX1 and CAT-I. Only CAT-I levels were increased by RO. CO and RMO may be a nutritional alternative to provide ALA for its bioconversion to EPA and DHA, and to increase the expression of PPAR-α, ACOX1 and CAT-I, especially CO-oil.

  7. Thermophilic Coenzyme B12-Dependent Acyl Coenzyme A (CoA) Mutase from Kyrpidia tusciae DSM 2912 Preferentially Catalyzes Isomerization of (R)-3-Hydroxybutyryl-CoA and 2-Hydroxyisobutyryl-CoA.

    PubMed

    Weichler, Maria-Teresa; Kurteva-Yaneva, Nadya; Przybylski, Denise; Schuster, Judith; Müller, Roland H; Harms, Hauke; Rohwerder, Thore

    2015-07-01

    The recent discovery of a coenzyme B12-dependent acyl-coenzyme A (acyl-CoA) mutase isomerizing 3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in the mesophilic bacterium Aquincola tertiaricarbonis L108 (N. Yaneva, J. Schuster, F. Schäfer, V. Lede, D. Przybylski, T. Paproth, H. Harms, R. H. Müller, and T. Rohwerder, J Biol Chem 287:15502-15511, 2012, http://dx.doi.org/10.1074/jbc.M111.314690) could pave the way for a complete biosynthesis route to the building block chemical 2-hydroxyisobutyric acid from renewable carbon. However, the enzyme catalyzes only the conversion of the stereoisomer (S)-3-hydroxybutyryl-CoA at reasonable rates, which seriously hampers an efficient combination of mutase and well-established bacterial poly-(R)-3-hydroxybutyrate (PHB) overflow metabolism. Here, we characterize a new 2-hydroxyisobutyryl-CoA mutase found in the thermophilic knallgas bacterium Kyrpidia tusciae DSM 2912. Reconstituted mutase subunits revealed highest activity at 55°C. Surprisingly, already at 30°C, isomerization of (R)-3-hydroxybutyryl-CoA was about 7,000 times more efficient than with the mutase from strain L108. The most striking structural difference between the two mutases, likely determining stereospecificity, is a replacement of active-site residue Asp found in strain L108 at position 117 with Val in the enzyme from strain DSM 2912, resulting in a reversed polarity at this binding site. Overall sequence comparison indicates that both enzymes descended from different prokaryotic thermophilic methylmalonyl-CoA mutases. Concomitant expression of PHB enzymes delivering (R)-3-hydroxybutyryl-CoA (beta-ketothiolase PhaA and acetoacetyl-CoA reductase PhaB from Cupriavidus necator) with the new mutase in Escherichia coli JM109 and BL21 strains incubated on gluconic acid at 37°C led to the production of 2-hydroxyisobutyric acid at maximal titers of 0.7 mM. Measures to improve production in E. coli, such as coexpression of the chaperone MeaH and repression of

  8. Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

    PubMed Central

    Hsiao, An-Shan; Xue, Yan

    2017-01-01

    Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

  9. Lipid membranes and acyl-CoA esters promote opposing effects on acyl-CoA binding protein structure and stability.

    PubMed

    Micheletto, Mariana C; Mendes, Luís F S; Basso, Luis G M; Fonseca-Maldonado, Raquel G; Costa-Filho, Antonio J

    2017-09-01

    Acyl-CoA Binding Proteins (ACBP) form a housekeeping family of proteins that is responsible for the buffering of long chain acyl-coenzyme A esters (LCFA-CoA) inside the cell. Even though numerous studies have focused on the characterization of different members of the ACBP family, the knowledge about the impact of both LCFA-CoA and phospholipids on ACBP structure and stability remains scarce. Besides, there are still controversies regarding the possible interaction of ACBP with biological membranes, even though this might be essential for the cargo capture and delivery. In this study, we observed that LCFA-CoA and phospholipids play opposite roles on protein stability and that the interaction with the membrane is dictated by electrostatic interaction. Furthermore, the results support the hypothesis that the LCFA-CoA delivery is driven by the increase of the negative charge on the membrane surface. The combined influence played by the different molecules on ACBP structure is discussed on the light of cargo capture/delivery giving new insights about this important process. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Developmental evolution in a patient with multiple acyl-coenzymeA dehydrogenase deficiency under pharmacological treatment.

    PubMed

    Rosa, M; Pascarella, A; Parenti, G; Buono, S; Romano, A; Della Casa, R; Andria, G; Marino, M; Riccio, M P; Bravaccio, C

    2012-03-01

    evaluate the psychomotor evolution of a child with Multiple acyl-CoA dehydrogenase deficiency after treatment with L-carnitine, ubiquinone and riboflavin. an assessment of psychomotor development was performed before the start of farmacological treatment using the Assessment Scale of Mental Development Griffiths (GMDS-R, 0-2 years). The same assessment was performed after a month and after six months of treatment to evaluate the possible benefits of treatment. we noticed a quick and dramatic improvement in muscular tone and motor performances after pharmacological treatment. We also observed a substantial improvement in the personal/social and hearing/language areas, suggesting the presence of intellectual/cognitive improvement. The clinical improvement correlated with the biochemical response. In our patient early therapy resulted in a optimal response in psychomotor development, motor function and muscole hypotonia. Evaluation with GMDS-R, a simple, non-invasive and multidimensional tool, represents a useful instrument to monitor the clinical response to treatment. Copyright © 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  11. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases. PMID:5500316

  12. Interactions of acyl-coenzyme A with phosphatidylcholine bilayers and serum albumin

    SciTech Connect

    Boylan, J.G.; Hamilton, J.A. )

    1992-01-21

    Interactions of oleoyl- and octanoyl-coenzyme A (CoA) with phosphatidylcholine (PC) vesicles and bovine serum albumin (BSA) were investigated by NMR spectroscopy. Binding of acyl-CoA to small unilamellar PC vesicles and to BSA was detected by changes in {sup 13}C and {sup 31}P chemical shifts relative to the chemical shifts for aqueous acyl-CoA. PC vesicles remained intact with {le} 15 mol % oleoyl-CoA, while higher oleoyl-CoA proportions produced mixed micelles. In contrast, {sup 13}C spectra revealed rapid exchange (ms) of octanoyl-CoA between the aqueous phase and PC vesicles and a low affinity for the bilayer. Thus, the binding affinity of acyl-CoA for PC bilayers is dependent on the acyl chain length. Addition of ({sup 13}C)carboxyl-enriched oleic acid to oleoyl-CoA/BSA mixtures revealed simultaneous binding of oleic acid and oleoyl-CoA to BSA, with some perturbation of binding interactions. Thus, BSA contains multiple binding sites for oleoyl-CoA and can bind fatty acid and acyl-CoA simultaneously.

  13. Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

    PubMed

    Whiteman, P A; Abraham, E P; Baldwin, J E; Fleming, M D; Schofield, C J; Sutherland, J D; Willis, A C

    1990-03-26

    A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

  14. Polyunsaturated fatty acyl-coenzyme As are inhibitors of cholesterol biosynthesis in zebrafish and mice

    PubMed Central

    Karanth, Santhosh; Tran, Vy My; Kuberan, Balagurunathan; Schlegel, Amnon

    2013-01-01

    SUMMARY Lipid disorders pose therapeutic challenges. Previously we discovered that mutation of the hepatocyte β-hydroxybutyrate transporter Slc16a6a in zebrafish causes hepatic steatosis during fasting, marked by increased hepatic triacylglycerol, but not cholesterol. This selective diversion of trapped ketogenic carbon atoms is surprising because acetate and acetoacetate can exit mitochondria and can be incorporated into both fatty acids and cholesterol in normal hepatocytes. To elucidate the mechanism of this selective diversion of carbon atoms to fatty acids, we fed wild-type and slc16a6a mutant animals high-protein ketogenic diets. We find that slc16a6a mutants have decreased activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), despite increased Hmgcr protein abundance and relative incorporation of mevalonate into cholesterol. These observations suggest the presence of an endogenous Hmgcr inhibitor. We took a candidate approach to identify such inhibitors. First, we found that mutant livers accumulate multiple polyunsaturated fatty acids (PUFAs) and PUFA-CoAs, and we showed that human HMGCR is inhibited by PUFA-CoAs in vitro. Second, we injected mice with an ethyl ester of the PUFA eicosapentaenoic acid and observed an acute decrease in hepatic Hmgcr activity, without alteration in Hmgcr protein abundance. These results elucidate a mechanism for PUFA-mediated cholesterol lowering through direct inhibition of Hmgcr. PMID:24057001

  15. Acyl-coenzyme A: cholesterol acyltransferase inhibitor Avasimibe affect survival and proliferation of glioma tumor cell lines.

    PubMed

    Bemlih, Sana; Poirier, Marie-Denise; El Andaloussi, Abdeljabar

    2010-06-15

    Glioblastoma is the most common primary brain tumor in adults and one of its hallmarks is resistance to apoptosis. Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular membrane-bound enzyme that uses cholesterol and long chain fatty acyl-CoA as substrates to produce cholesteryl esters. The presence of cholesteryl esters in glioblastoma may be related to vascular and/or cell neoplastic proliferation in the tumor mass, two prerequisites for tumor cell growth. ACAT activity has been detected in glioblastoma cell homogenates. The present study is the first report on the effect of Avasimibe, a specific inhibitor of ACAT, on glioma cell lines (U87, A172 and GL261). Our results showed that Avasimibe inhibited ACAT-1 expression and cholesterol ester synthesis in glioma cell lines. Moreover, Avasimibe inhibited the growth of the cells by inducing cell cycle arrest and induced apoptosis as a result of caspase-8 and caspase-3 activation. Also, Our findings provide proof of principle that targeting ACAT-1 with the inhibitor Avasimibe could be an efficient therapy in the treatment of glioblastoma.

  16. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    SciTech Connect

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  17. Impact of subdermal norgestrel on hepatic acyl-coenzyme A:cholesterol- acyltransferase (ACAT) activity: possible antiatherogenic effect.

    PubMed

    Letterie, G S

    2000-06-01

    The impact of subdermally placed ethinyl estradiol, norgestrel, and the combination of the two on cholesterol metabolism as measured by hepatic acyl:cholesterol-acyltransferase (ACAT) activity was examined in the rat model. A total of 48 rats were assigned to one of 6 groups, receiving either 0.1 mg or 1.0 mg of ethinyl estradiol daily, 1.0 or 10 mg of norgestrel daily, and combinations of either 0.1 mg ethinyl estradiol/1.0 mg norgestrel or 1.0 mg ethinyl estradiol/10 mg norgestrel daily. All drugs were administered through subdermally placed time release capsules. The administration of norgestrel only in either 1.0 mg or 10 mg resulted in significantly lower rates of ACAT activity (0.77 +/- 0.566 and 0.91 +/- 0.239 pmol/mg/min, respectively). The combination of 1.0 ethinyl estradiol and 10 mg norgestrel resulted in a significant increase in ACAT activity to 2.17 +/- 0.873. This combination also resulted in significantly greater weight loss at the conclusion of treatment [247.83 +/- 6.2 g (pre) vs. 205.50 +/- 10.6 (post)]. There were no other differences in ACAT activity between groups and no other differences in weight, both between groups and pre- and post-treatment within groups. In summary, subdermally placed norgestrel resulted in a significant lowering of ACAT activity not seen with either administration of ethinyl estradiol alone or the combination of ethinyl estradiol and norgestrel in doses ranging from 0.1 to 1.0 mg of ethinyl estradiol and 1.0 to 10.0 mg of norgestrel. Significantly increased ACAT activity for the combination of 1.0 ethinyl estradiol and 10 mg norgestrel over either ethinyl estradiol or norgestrel alone or a lower dose combination suggests a dose-related threshold and drug-drug interaction for this effect. These results suggest that subdermally placed norgestrel may result in significantly lower ACAT activity and may have a potential role as an antiatherogenic treatment.

  18. Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

    PubMed

    Aznar-Moreno, Jose A; Venegas Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Mullen, Robert; Gidda, Satinder K; Salas, Joaquín J

    2014-03-01

    Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.

  19. Continuous recording of long-chain acyl-coenzyme a synthetase activity using fluorescently labeled bovine serum albumin.

    PubMed

    Demant, E J; Nystrøm, B T

    2001-08-01

    The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2-0.4 microM) are 5-10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 microM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.

  20. Inhibition of Acyl-Coenzyme A:Cholesterol Acyltransferase 2 (ACAT2) Prevents Dietary Cholesterol-associated Steatosis by Enhancing Hepatic Triglyceride Mobilization*

    PubMed Central

    Alger, Heather M.; Brown, J. Mark; Sawyer, Janet K.; Kelley, Kathryn L.; Shah, Ramesh; Wilson, Martha D.; Willingham, Mark C.; Rudel, Lawrence L.

    2010-01-01

    Acyl-CoA:cholesterol O-acyl transferase 2 (ACAT2) promotes cholesterol absorption by the intestine and the secretion of cholesteryl ester-enriched very low density lipoproteins by the liver. Paradoxically, mice lacking ACAT2 also exhibit mild hypertriglyceridemia. The present study addresses the unexpected role of ACAT2 in regulation of hepatic triglyceride (TG) metabolism. Mouse models of either complete genetic deficiency or pharmacological inhibition of ACAT2 were fed low fat diets containing various amounts of cholesterol to induce hepatic steatosis. Mice genetically lacking ACAT2 in both the intestine and the liver were dramatically protected against hepatic neutral lipid (TG and cholesteryl ester) accumulation, with the greatest differences occurring in situations where dietary cholesterol was elevated. Further studies demonstrated that liver-specific depletion of ACAT2 with antisense oligonucleotides prevents dietary cholesterol-associated hepatic steatosis both in an inbred mouse model of non-alcoholic fatty liver disease (SJL/J) and in a humanized hyperlipidemic mouse model (LDLr−/−, apoB100/100). All mouse models of diminished ACAT2 function showed lowered hepatic triglyceride concentrations and higher plasma triglycerides secondary to increased hepatic secretion of TG into nascent very low density lipoproteins. This work demonstrates that inhibition of hepatic ACAT2 can prevent dietary cholesterol-driven hepatic steatosis in mice. These data provide the first evidence to suggest that ACAT2-specific inhibitors may hold unexpected therapeutic potential to treat both atherosclerosis and non-alcoholic fatty liver disease. PMID:20231283

  1. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

    PubMed

    Chen, Wei-Ching; Wang, Chih-Yang; Hung, Yu-Hsuan; Weng, Tzu-Yang; Yen, Meng-Chi; Lai, Ming-Derg

    2016-01-01

    Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers.

  2. Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase.

    PubMed

    Kim, Ji-Hyun; Lee, Hyo-Jung; Jeong, Soo-Jin; Lee, Min-Ho; Kim, Sung-Hoon

    2012-09-01

    Hyperlipidemia is an important factor to induce metabolic syndrome such as obesity, diabetes and cardiovascular diseases. Recently, some antihyperlipidemic agents from herbal medicines have been in the spotlight in the medical science field. Thus, the present study evaluated the antihyperlipidemic activities of the essential oil from the leaves of Pinus koraiensis SIEB (EOPK) that has been used as a folk remedy for heart disease. The reverse transcription polymerase chain reaction (RT-PCR) revealed that EOPK up-regulated low density lipoprotein receptor (LDLR) at the mRNA level as well as negatively suppressed the expression of sterol regulatory element-binding protein (SREBP)-1c, SREBP-2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) involved in lipid metabolism in HepG2 cells. Also, western blotting showed that EOPK activated LDLR and attenuated the expression of FAS at the protein level in the cells. Consistently, EOPK significantly inhibited the level of human acylcoenzyme A: cholesterol acyltransferase (hACAT)1 and 2 and reduced the low-density lipoprotein (LDL) oxidation activity. Furthermore, chromatography-mass spectrometry (GC-MS) analysis showed that EOPK, an essential oil mixture, contained camphene (21.11%), d-limonene (21.01%), α-pinene (16.74%) and borneol (11.52%). Overall, the findings suggest that EOPK can be a potent pharmaceutical agent for the prevention and treatment of hyperlipidemia.

  3. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/sterol carrier protein-x gene function

    PubMed Central

    Seedorf, Udo; Raabe, Martin; Ellinghaus, Peter; Kannenberg, Frank; Fobker, Manfred; Engel, Thomas; Denis, Simone; Wouters, Fred; Wirtz, Karel W.A.; Wanders, Ronald J.A.; Maeda, Nobuyo; Assmann, Gerd

    1998-01-01

    Gene targeting in mice was used to investigate the unknown function of Scp2, encoding sterol carrier protein-2 (SCP2; a peroxisomal lipid carrier) and sterol carrier protein-x (SCPx; a fusion protein between SCP2 and a peroxisomal thiolase). Complete deficiency of SCP2 and SCPx was associated with marked alterations in gene expression, peroxisome proliferation, hypolipidemia, impaired body weight control, and neuropathy. Along with these abnormalities, catabolism of methyl-branched fatty acyl CoAs was impaired. The defect became evident from up to 10-fold accumulation of the tetramethyl-branched fatty acid phytanic acid in Scp2(−/−) mice. Further characterization supported that the gene disruption led to inefficient import of phytanoyl-CoA into peroxisomes and to defective thiolytic cleavage of 3-ketopristanoyl-CoA. These results corresponded to high-affinity binding of phytanoyl-CoA to the recombinant rat SCP2 protein, as well as high 3-ketopristanoyl-CoA thiolase activity of the recombinant rat SCPx protein. PMID:9553048

  4. Wax Ester Production from n-Alkanes by Acinetobacter sp. Strain M-1: Ultrastructure of Cellular Inclusions and Role of Acyl Coenzyme A Reductase

    PubMed Central

    Ishige, Takeru; Tani, Akio; Takabe, Keiji; Kawasaki, Kazunori; Sakai, Yasuyoshi; Kato, Nobuo

    2002-01-01

    Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis. PMID:11872467

  5. Identification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis.

    PubMed

    Li, Fengling; Wu, Xuemin; Lam, Patricia; Bird, David; Zheng, Huanquan; Samuels, Lacey; Jetter, Reinhard; Kunst, Ljerka

    2008-09-01

    Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C(16) and C(18)) or very-long-chain (C(20) and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.

  6. Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer

    PubMed Central

    Chen, Wei-Ching; Wang, Chih-Yang; Hung, Yu-Hsuan; Weng, Tzu-Yang; Yen, Meng-Chi; Lai, Ming-Derg

    2016-01-01

    Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers. PMID:27171439

  7. The Effects of Thyroid Hormones on Gene Expression of Acyl-Coenzyme A Thioesterases in Adipose Tissue and Liver of Mice.

    PubMed

    Krause, Kerstin; Weiner, Juliane; Hönes, Sebastian; Klöting, Nora; Rijntjes, Eddy; Heiker, John T; Gebhardt, Claudia; Köhrle, Josef; Führer, Dagmar; Steinhoff, Karen; Hesse, Swen; Moeller, Lars C; Tönjes, Anke

    2015-09-01

    Thyroid hormones (TH) exert pleiotropic effects on glucose and lipid homeostasis. However, it is as yet unclear how TH regulate lipid storage and utilization in order to adapt to metabolic needs. Acyl-CoA thioesterases (ACOTs) have been proposed to play a regulatory role in the metabolism of fatty acids. We investigated the interaction between thyroid dysfunction and Acot expression in adipose tissues and livers of thyrotoxic and hypothyroid mice. Ten-week-old female C57BL/6NTac mice (n = 10/group) were made hyperthyroid by the application of L-thyroxine (2 µg/ml in drinking water) for 4 weeks. Hypothyroidism was induced in 10-week-old mice by feeding an iodine-free chow supplemented with 0.15% PTU for 4 weeks. We measured mRNA expression levels of Acot8, 11 and 13 in the liver and epididymal and inguinal white and brown adipose tissues (BAT). Furthermore, we investigated hepatic Acot gene expression in TRα- and TRβ-deficient mice. We showed that the expression of Acot8, 11 and 13 is predominantly stimulated by a thyrotoxic state in the epididymal white adipose tissue. In contrast, hypothyroidism predominantly induces the expression of Acot8 in BAT in comparison with BAT of thyrotoxic and euthyroid mice (p < 0.01). However, no significant changes in Acot expression were observed in inguinal white adipose tissue. In liver, Acot gene expression is collectively elicited by a thyrotoxic state. These data suggest that ACOTs are targets of TH and are likely to influence 3,5,3'-triiodo-L-thyronine-orchestrated mechanisms of lipid uptake, storage and utilization to adapt the regulation of metabolic demands.

  8. Identification of the Wax Ester Synthase/Acyl-Coenzyme A:Diacylglycerol Acyltransferase WSD1 Required for Stem Wax Ester Biosynthesis in Arabidopsis12[W][OA

    PubMed Central

    Li, Fengling; Wu, Xuemin; Lam, Patricia; Bird, David; Zheng, Huanquan; Samuels, Lacey; Jetter, Reinhard; Kunst, Ljerka

    2008-01-01

    Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C16 and C18) or very-long-chain (C20 and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment. PMID:18621978

  9. Characterization of Staphylococcus aureus SarA binding sites.

    PubMed

    Sterba, Kristen M; Mackintosh, Samuel G; Blevins, Jon S; Hurlburt, Barry K; Smeltzer, Mark S

    2003-08-01

    The staphylococcal accessory regulator locus (sarA) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and cis elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene cna. The results suggest the existence of multiple high-affinity binding sites within the cna promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including cna, fnbA, and sspA. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the Staphylococcus aureus genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the S. aureus genes spa, fnbA, sspA, agr, hla, and cna. With the exception of cna, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.

  10. Acyl-CoA binding proteins: multiplicity and function.

    PubMed

    Gossett, R E; Frolov, A A; Roths, J B; Behnke, W D; Kier, A B; Schroeder, F

    1996-09-01

    The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

  11. Scatchard analysis of fluorescent concanavalin A binding to lymphocytes

    SciTech Connect

    Gordon, I.L.

    1995-07-01

    Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothio-cyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4{degrees}C was compared to {sup 125}I-ConA binding. A FACS IV flow cytometer was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer-determined fluorescence and ligand binding per cell. As FITC-ConA binding showed a quasi-Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC-ConA binding yielded results (1.9 x 10{sup 6} receptors/cell, K = 3.6 x 10{sup -15}) similar to those obtained With {sup 125}I-ConA (1.4 x 10{sup 6} receptors/cell, K = 5.2 x 10{sup -15}). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. 32 refs., 4 figs., 1 tab.

  12. Fibrinopeptide A binds Gly-Pro-Arg-Pro.

    PubMed Central

    Root-Bernstein, R S; Westall, F C

    1984-01-01

    The tetrapeptide Gly-Pro-Arg-Pro inhibits fibrinogen aggregation, probably by binding to the same sites used during initiation of fibrin formation. The Gly-Pro-Arg-Pro binding sites have not yet been identified. However, their possible sequence and locations have been predicted on the basis of the amino acid pairing hypothesis. One of these predicted sites is on fibrinopeptide A. We report here that nuclear magnetic resonance studies indicate that Gly-Pro-Arg-Pro binds to fibrinopeptide A with a binding constant, K, of ca. 10(4) per mol. We also report results of 19 related peptide combinations used as controls. PMID:6589598

  13. Inhibition of tristetraprolin deadenylation by poly(A) binding protein

    PubMed Central

    Rowlett, Robert M.; Chrestensen, Carol A.; Schroeder, Melanie J.; Harp, Mary G.; Pelo, Jared W.; Shabanowitz, Jeffery; DeRose, Robert; Hunt, Donald F.; Sturgill, Thomas W.; Worthington, Mark T.

    2008-01-01

    Tristetraprolin (TTP) is the prototype for a family of RNA binding proteins that bind the tumor necrosis factor (TNF) messenger RNA AU-rich element (ARE), causing deadenylation of the TNF poly(A) tail, RNA decay, and silencing of TNF protein production. Using mass spectrometry sequencing we identified poly(A) binding proteins-1 and -4 (PABP1 and PABP4) in high abundance and good protein coverage from TTP immunoprecipitates. PABP1 significantly enhanced TNF ARE binding by RNA EMSA and prevented TTP-initiated deadenylation in an in vitro macrophage assay of TNF poly(A) stability. Neomycin inhibited TTP-promoted deadenylation at concentrations shown to inhibit the deadenylases poly(A) ribonuclease and CCR4. Stably transfected RAW264.7 macrophages overexpressing PABP1 do not oversecrete TNF; instead they upregulate TTP protein without increasing TNF protein production. The PABP1 inhibition of deadenylation initiated by TTP does not require the poly(A) binding regions in RRM1 and RRM2, suggesting a more complicated interaction than simple masking of the poly(A) tail from a 3′-exonuclease. Like TTP, PABP1 is a substrate for p38 MAP kinase. Finally, PABP1 stabilizes cotransfected TTP in 293T cells and prevents the decrease in TTP levels seen with p38 MAP kinase inhibition. These findings suggest several levels of functional antagonism between TTP and PABP1 that have implications for regulation of unstable mRNAs like TNF. PMID:18467502

  14. Vaccine-Elicited 10-Kilodalton Culture Filtrate Protein-Specific CD8+ T Cells Are Sufficient To Mediate Protection against Mycobacterium tuberculosis Infection▿

    PubMed Central

    Wu, Ying; Woodworth, Joshua S.; Shin, Daniel S.; Morris, Sheldon; Behar, Samuel M.

    2008-01-01

    The 10-kDa culture filtrate protein (CFP-10) and 6-kDa early secretory antigen of T cells (ESAT-6) are secreted in abundance by Mycobacterium tuberculosis and are frequently recognized by T cells from infected people. The genes encoding these proteins have been deleted from the genome of the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG), and it is hypothesized that these proteins are important targets of protective immunity. Indeed, vaccination with ESAT-6 elicits protective CD4+ T cells in C57BL/6 mice. We have previously shown that M. tuberculosis infection of C3H mice elicits CFP-10-specific CD8+ and CD4+ T cells. Here we demonstrate that immunization with a CFP-10 DNA vaccine stimulates a specific T-cell response only to the H-2Kk-restricted epitope CFP-1032-39. These CFP-1032-39-specific CD8+ cells undergo a rapid expansion and accumulate in the lung following challenge of immunized mice with aerosolized M. tuberculosis. Protective immunity is induced by CFP-10 DNA vaccination as measured by a CFU reduction in the lung and spleen 4 and 8 weeks after challenge with M. tuberculosis. These data demonstrate that CFP-10 is a protective antigen and that CFP-1032-39-specific CD8+ T cells elicited by vaccination are sufficient to mediate protection against tuberculosis. PMID:18332205

  15. Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts.

    PubMed Central

    Launois, P; N'Diaye, M N; Cartel, J L; Mane, I; Drowart, A; Van Vooren, J P; Sarthou, J L; Huygen, K

    1995-01-01

    Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and interleukin-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine. PMID:7806388

  16. Evaluation of the Recombinant 10-Kilodalton Immunodominant Region of the BP26 Protein of Brucella abortus for Specific Diagnosis of Bovine Brucellosis ▿

    PubMed Central

    Tiwari, Arvind Kumar; Kumar, Subodh; Pal, Vijai; Bhardwaj, Bhupendra; Rai, Ganga Prasad

    2011-01-01

    Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohen's kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals. PMID:21852548

  17. The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

    PubMed

    Koetsier, Martijn J; Gombert, Andreas K; Fekken, Susan; Bovenberg, Roel A L; van den Berg, Marco A; Kiel, Jan A K W; Jekel, Peter A; Janssen, Dick B; Pronk, Jack T; van der Klei, Ida J; Daran, Jean-Marc

    2010-01-01

    Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

  18. Evolution of the acyl-CoA binding protein (ACBP)

    PubMed Central

    Burton, Mark; Rose, Timothy M.; Færgeman, Nils J.; Knudsen, Jens

    2005-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein that binds C12–C22 acyl-CoA esters with high affinity. In vitro and in vivo experiments suggest that it is involved in multiple cellular tasks including modulation of fatty acid biosynthesis, enzyme regulation, regulation of the intracellular acyl-CoA pool size, donation of acyl-CoA esters for β-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified in all four eukaryotic kingdoms, Animalia, Plantae, Fungi and Protista, and eleven eubacterial species. ACBP homologues were not detected in any other known bacterial species, or in archaea. Nearly all of the ACBP-containing bacteria are pathogenic to plants or animals, suggesting that an ACBP gene could have been acquired from a eukaryotic host by horizontal gene transfer. Many bacterial, fungal and higher eukaryotic species only harbour a single ACBP homologue. However, a number of species, ranging from protozoa to vertebrates, have evolved two to six lineage-specific paralogues through gene duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage-specific paralogues have evolved altered functions. The appearance of ACBP very early on in evolution points towards a fundamental role of ACBP in acyl-CoA metabolism, including ceramide synthesis and in signalling. PMID:16018771

  19. A Binding Domain on Mesothelin for CA125/MUC16*

    PubMed Central

    Kaneko, Osamu; Gong, Lucy; Zhang, Jingli; Hansen, Johanna K.; Hassan, Raffit; Lee, Byungkook; Ho, Mitchell

    2009-01-01

    Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296–359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296–359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors. PMID:19075018

  20. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

    PubMed Central

    Henry, Kevin A.; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O.; MacKenzie, C. Roger; Arbabi-Ghahroudi, Mehdi

    2016-01-01

    Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. PMID:27631624

  1. Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus.

    PubMed

    Raboanatahiry, Nadia Haingotiana; Lu, Guangyuan; Li, Maoteng

    2015-01-01

    Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.

  2. The Verrucomicrobia LexA-Binding Motif: Insights into the Evolutionary Dynamics of the SOS Response

    PubMed Central

    Erill, Ivan; Campoy, Susana; Kılıç, Sefa; Barbé, Jordi

    2016-01-01

    The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls. PMID:27489856

  3. Superovulation alters embryonic poly(A)-binding protein (Epab) and poly(A)-binding protein, cytoplasmic 1 (Pabpc1) gene expression in mouse oocytes and early embryos.

    PubMed

    Ozturk, Saffet; Yaba-Ucar, Aylin; Sozen, Berna; Mutlu, Derya; Demir, Necdet

    2016-03-01

    Embryonic poly(A)-binding protein (EPAB) and poly(A)-binding protein, cytoplasmic 1 (PABPC1) play critical roles in translational regulation of stored maternal mRNAs required for proper oocyte maturation and early embryo development in mammals. Superovulation is a commonly used technique to obtain a great number of oocytes in the same developmental stages in assisted reproductive technology (ART) and in clinical or experimental animal studies. Previous studies have convincingly indicated that superovulation alone can cause impaired oocyte maturation, delayed embryo development, decreased implantation rate and increased postimplantation loss. Although how superovulation results in these disturbances has not been clearly addressed yet, putative changes in genes related to oocyte and early embryo development seem to be potential risk factors. Thus, the aim of the present study was to determine the effect of superovulation on Epab and Pabpc1 gene expression. To this end, low- (5IU) and high-dose (10IU) pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotrophin (hCG) were administered to female mice to induce superovulation, with naturally cycling female mice serving as controls. Epab and Pabpc1 gene expression in germinal vesicle (GV) stage oocytes, MII oocytes and 1- and 2-cell embryos collected from each group were quantified using quantitative reverse transcription-polymerase chain reaction. Superovulation with low or high doses of gonadotropins significantly altered Epab and Pabpc1 mRNA levels in GV oocytes, MII oocytes and 1- and 2-cell embryos compared with their respective controls (P<0.05). These changes most likely lead to variations in expression of EPAB- and PABPC1-regulated genes, which may adversely influence the quality of oocytes and early embryos retrieved using superovulation.

  4. Structures of Triacetyloleandomycin and Mycalamide A Bind to the Large Ribosomal Subunit of Haloarcula marismortui

    SciTech Connect

    Gürel, Güliz; Blaha, Gregor; Steitz, Thomas A.; Moore, Peter B.

    2010-01-14

    Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the large ribosomal subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.

  5. A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere

    PubMed Central

    Lo, Anthony W.I.; Craig, Jeffrey M.; Saffery, Richard; Kalitsis, Paul; Irvine, Danielle V.; Earle, Elizabeth; Magliano, Dianna J.; Choo, K.H.Andy

    2001-01-01

    Centromere protein A (CENP-A) is an essential centromere-specific histone H3 homologue. Using combined chromatin immunoprecipitation and DNA array analysis, we have defined a 330 kb CENP-A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10). This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through lower-resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP-A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP-A within centromere-specific nucleosomes. The DNA within this domain has a high AT-content comparable to that of α-satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid-S phase. Neocentromere formation is accompanied by a replication time lag around but not within the CENP-A binding region, with this lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres. PMID:11296241

  6. Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein.

    PubMed

    Leung, Ka-Chun; Li, Hong-Ye; Xiao, Shi; Tse, Muk-Hei; Chye, Mee-Len

    2006-04-01

    Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs) function in the storage and intracellular transport of acyl-CoA esters in eukaryotes. Fatty acids synthesized de novo in plant chloroplasts are exported as oleoyl-CoA and palmitoyl-CoA esters. In Arabidopsis, other than the 10-kDa ACBP, there exists five larger ACBPs (ACBP1 to ACBP5) of which homologues have not been characterized in other organisms. To investigate the significance of this gene family, we have attempted to subcellularly localize them and compare their acyl-CoA-binding affinities. We have previously shown that Arabidopsis ACBP1 and ACBP2 are membrane-associated proteins while ACBP4 and ACBP5 contain kelch motifs. Here, to localize ACBP3, we have expressed ACBP3-red fluorescent protein (DsRed2) from the CaMV 35S promoter. ACBP3-DsRed was localized extracellularly in transiently expressed tobacco BY-2 cells and onion epidermal cells. The function of the acyl-CoA-binding domain in ACBP3 was investigated by in vitro binding assays using (His)(6)-ACBP3, which was observed to bind [(14)C]arachidonyl-CoA with high affinity in comparison to [(14)C]palmitoyl-CoA and [(14)C]oleoyl-CoA. To identify the residues functional in binding, five mutants with single amino acid substitutions in the acyl-CoA-binding domain of (His)(6)-ACBP3 and (His)(6)-ACBP1 (which also binds [(14)C]arachidonyl-CoA) were generated by site-directed mutagenesis. Binding assays with arachidonyl-CoA revealed that replacement of a conserved R residue (R150A in ACBP1 and R284A in ACBP3), disrupted binding. In contrast, other substitutions in ACBP1 (Y126A, K130A, K152A and Y171A) and in ACBP3 (F260A, K264A, K286A and Y305A) did not affect arachidonyl-CoA binding, unlike their equivalents in (His)(6)-ACBP2, (His)(6)-ACBP4 and (His)(6)-ACBP5, which had altered binding to palmitoyl-CoA or oleoyl-CoA.

  7. Increased /sup 125/I-labelled concanavalin A binding to erythrocytes in diabetes mellitus

    SciTech Connect

    Okada, Y.; Arima, T.; Okazaki, S.; Nakata, K.; Nagashima, H.; Yamabuki, T.

    1982-03-01

    Percentage binding of /sup 125/I-labelled concanavalin A to erythrocytes in diabetic patients was significantly higher than that in normal subjects (12.2 +- 2.8 versus 8.1 +- 1.8%, mean +- SD, p < 0.001). Insulin-dependent diabetic patients showed significantly higher concanavalin A binding than non-insulin-dependent diabetic subjects (15.0 +- 1.4 versus 11.4 +- 2.5%, p < 0.01). There was a highly significant correlation between percentage binding of /sup 125/I-labelled concanavalin A and glycosylated haemoglobin.

  8. The Xenopus laevis ribosomal gene promoter contains a binding site for nuclear factor-1.

    PubMed Central

    Walker, P; Reeder, R H

    1988-01-01

    Nuclear Factor I (NF1) is a DNA binding protein that is known to function in the replication of Adeno virus and also binds to many promoters recognized by RNA polymerase II. We have found that there is also an NF1 binding site within the ribosomal gene promoter from Xenopus laevis as well as in several other promoters recognized by RNA polymerase I. The function of a binding site for a polymerase II transcription factor within a promoter recognized by polymerase I is not known. However, its presence suggests interesting regulatory possibilities. Images PMID:3205719

  9. An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

    PubMed Central

    Sanchez-Alberola, Neus; Campoy, Susana; Emerson, David; Barbé, Jordi

    2015-01-01

    ABSTRACT The SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In the Betaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacterium Sideroxydans lithotrophicus. In silico and in vitro analyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of the Bacillus subtilis LexA-binding motif. We confirm that the closely related Gallionella capsiferriformans shares the same LexA-binding motif, and in silico analyses indicate that this motif is also conserved in the Nitrosomonadales and the Methylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within the Betaproteobacteria. However, their lexA gene is unrelated to the standard Betaproteobacteria lexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon of S. lithotrophicus is comparable in size and function to that of many other Betaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain of S. lithotrophicus LexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make the B. subtilis-type motif an optimal interface for multiple LexA sequences. IMPORTANCE Understanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or

  10. Identification of FAM96B as a novel prelamin A binding partner

    SciTech Connect

    Xiong, Xing-Dong; Wang, Junwen; Zheng, Huiling; Jing, Xia; Liu, Zhenjie; Zhou, Zhongjun; Liu, Xinguang

    2013-10-11

    Highlights: •We screen the binding protein of prelamin A by yeast two-hybrid screen. •FAM96B colocalizes with prelamin A in HEK-293 cells. •FAM96B physically interacts with prelamin A. -- Abstract: Prelamin A accumulation causes nuclear abnormalities, impairs nuclear functions, and eventually promotes cellular senescence. However, the underlying mechanism of how prelamin A promotes cellular senescence is still poorly understood. Here we carried out a yeast two-hybrid screen using a human skeletal muscle cDNA library to search for prelamin A binding partners, and identified FAM96B as a prelamin A binding partner. The interaction of FAM96B with prelamin A was confirmed by GST pull-down and co-immunoprecipitation experiments. Furthermore, co-localization experiments by fluorescent confocal microscopy revealed that FAM96B colocalized with prelamin A in HEK-293 cells. Taken together, our data demonstrated the physical interaction between FAM96B and prelamin A, which may provide some clues to the mechanisms of prelamin A in premature aging.

  11. Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony

    PubMed Central

    Jha, Jyoti K.

    2016-01-01

    The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites. The altered SeqA binding, however, does not affect initiation synchrony. Synchrony is also unaffected when individual SeqA sites are mutated. An apparent exception was one mutant where the mutation also changed an overlapping DnaA site. In this mutant, the observed asynchrony could be from altered DnaA binding, as selectively mutating this SeqA site did not cause asynchrony. These results reveal robust initiation synchrony against alterations of individual SeqA binding sites. The redundancy apparently ensures SeqA function in controlling replication in E. coli. PMID:27930658

  12. Results on a binding neuron model and their implications for modified hourglass model for neuronal network.

    PubMed

    Arunachalam, Viswanathan; Akhavan-Tabatabaei, Raha; Lopez, Cristina

    2013-01-01

    The classical models of single neuron like Hodgkin-Huxley point neuron or leaky integrate and fire neuron assume the influence of postsynaptic potentials to last till the neuron fires. Vidybida (2008) in a refreshing departure has proposed models for binding neurons in which the trace of an input is remembered only for a finite fixed period of time after which it is forgotten. The binding neurons conform to the behaviour of real neurons and are applicable in constructing fast recurrent networks for computer modeling. This paper develops explicitly several useful results for a binding neuron like the firing time distribution and other statistical characteristics. We also discuss the applicability of the developed results in constructing a modified hourglass network model in which there are interconnected neurons with excitatory as well as inhibitory inputs. Limited simulation results of the hourglass network are presented.

  13. Studies on the specificity of the IgA-binding lectin, jacalin.

    PubMed

    Skea, D L; Christopoulous, P; Plaut, A G; Underdown, B J

    1988-01-01

    The interactions of IgA with the jackfruit lectin, jacalin, were investigated with regard to the specificity of jacalin for species and subclasses of IgA. It was found that jacalin selectively bound to human IgA1, but not to human IgA2, mouse IgA or rat IgA. Binding studies with human IgA1 fragments produced by different IgA1 proteases revealed that jacalin bound to galactose-terminal oligosaccharides in the hinge region of human IgA1. Affinity chromatography employing jacalin-Sepharose provided a means to separate the subclasses of IgA in human whey.

  14. Polyalanine-independent conformational conversion of nuclear poly(A)-binding protein 1 (PABPN1).

    PubMed

    Winter, Reno; Kühn, Uwe; Hause, Gerd; Schwarz, Elisabeth

    2012-06-29

    Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.

  15. Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

    PubMed

    Prochnow, D; Riedel, N; Agutter, P S; Fasold, H

    1990-04-25

    We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).

  16. An Arabidopsis family of six acyl-CoA-binding proteins has three cytosolic members.

    PubMed

    Xiao, Shi; Chye, Mee-Len

    2009-06-01

    In Arabidopsis thaliana, a gene family of six members encodes acyl-CoA-binding proteins (ACBPs). These Arabidopsis ACBPs (designated ACBP1 to ACBP6) range in size from 10.4kDa to 73.1kDa and display varying affinities for acyl-CoA esters, suggesting that they have different roles in plant lipid metabolism. In contrast, only the 10-kDa ACBPs have been well-characterized from other eukaryote species. Our previous studies have revealed that ACBP1 and ACBP2 are membrane-associated proteins, while ACBP3 is extracellularly-targeted. More recently, we have reported that the remaining three members in this protein family (namely ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol in Arabidopsis. The subcellular localizations of ACBP4, ACBP5 and ACBP6 in the cytosol were demonstrated using a number of different approaches incorporating biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immunoelectron microscopy using ACBP-specific antibodies. Our results indicate that all three ACBPs in the cytosol are potential candidates for acyl-CoA binding and trafficking in plant cells. In this review, the functional redundancy and differences among the three cytosolic ACBPs are discussed by comparison of their light-regulated expression and substrate affinities to acyl-CoA esters, and from biochemical analyses on their knockout mutants and/or overexpression in transgenic Arabidopsis. The transcriptionally light-induced ACBP4 and ACBP5, which encode the two largest forms of Arabidopsis ACBPs, bind oleoyl-CoA esters and likely transfer oleoyl-CoAs from the plastids (the site of de novo fatty acid biosynthesis) to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids in Arabidopsis.

  17. Characterization and quantitation of concanavalin A binding by plasma membrane enriched fractions from soybean root

    SciTech Connect

    Berkowitz, R.L.; Travis, R.L.

    1981-11-01

    The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritated (/sup 3/H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of /sup 3/H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10/sup 15/ molecules of /sup 3/H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of ..cap alpha..-methyl mannoside. A major peak of /sup 3/H-Con A binding was also observed in fractions recovered from the 25/30% interface of a complex discontinuous sucrose density gradient when membranes were isolated in the absence of Mg/sup 2 +/. When high Mg/sup 2 +/ was present in the isolation and gradient media, the peak was shifted to a fraction recovered from the 34/38% sucrose interface. These results suggest that Con A binding sites are also present on membranes of the endoplasmic reticulum. The amount of Con A bound by endoplasmic reticulum membranes was at least twice the amount bound by membranes in plasma membrane enriched fractions when binding was compared on a per unit membrane protein basis. In contrast, mitochondrial inner membranes, which equilibrate at the same density as plasma membranes, had little ability to bind the lectin.

  18. Elevated brain monoamine oxidase A binding in the early postpartum period.

    PubMed

    Sacher, Julia; Wilson, Alan A; Houle, Sylvain; Rusjan, Pablo; Hassan, Sabrina; Bloomfield, Peter M; Stewart, Donna E; Meyer, Jeffrey H

    2010-05-01

    The early postpartum period is a time of high risk for a major depressive episode (or postpartum depression), with a prevalence of 13%. During this time, there is a heightened vulnerability for low mood because postpartum blues is common. Severe postpartum blues can herald the onset of postpartum depression. The neurobiological mechanisms to explain postpartum blues and the high risk for the onset of postpartum depression in the first few weeks after delivery are unclear. Estrogen levels drop 100- to 1000-fold during the first 3 to 4 days postpartum, and changes in estrogen levels have an inverse relationship with monoamine oxidase A (MAO-A) density. However, MAO-A levels have never been measured in the early postpartum period. To determine whether brain MAO-A binding is elevated in the early postpartum period. Case-control study. Tertiary care academic psychiatric hospital in Toronto, Ontario, Canada. Fifteen healthy women who were 4 to 6 days postpartum and 15 healthy women who had not recently been postpartum underwent carbon 11-labeled harmine positron emission tomography scanning. All women were nonsmoking and medication free. MAO-A total distribution volume, an index of MAO-A density, was measured in prefrontal cortex, anterior cingulate cortex, anterior temporal cortex, thalamus, dorsal putamen, hippocampus, and midbrain. MAO-A total distribution volume was significantly elevated (mean, 43%) throughout all analyzed brain regions during the early postpartum period. Elevated MAO-A levels in the early postpartum period can be interpreted as a marker of a monoamine-lowering process that contributes to the mood change of postpartum blues. Rather than a purely psychosocial model, we propose a neurobiological model of estrogen decline, followed by elevated MAO-A binding, low mood, and subsequently a period of high risk for major depressive episodes. Our model has important implications for preventing postpartum depression and for developing therapeutic strategies

  19. Lin28A binds active promoters and recruits Tet1 to regulate gene expression

    PubMed Central

    Zeng, Yaxue; Yao, Bing; Shin, Jaehoon; Lin, Li; Kim, Namshik; Song, Qifeng; Liu, Shuang; Su, Yijing; Guo, Junjie U.; Huang, Luoxiu; Wan, Jun; Wu, Hao; Qian, Jiang; Cheng, Xiaodong; Zhu, Heng; Ming, Guo-li; Jin, Peng; Song, Hongjun

    2015-01-01

    Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites, and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems. PMID:26711009

  20. Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression.

    PubMed

    Zeng, Yaxue; Yao, Bing; Shin, Jaehoon; Lin, Li; Kim, Namshik; Song, Qifeng; Liu, Shuang; Su, Yijing; Guo, Junjie U; Huang, Luoxiu; Wan, Jun; Wu, Hao; Qian, Jiang; Cheng, Xiaodong; Zhu, Heng; Ming, Guo-li; Jin, Peng; Song, Hongjun

    2016-01-07

    Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Cubilin, a binding partner for galectin-3 in the murine utero-placental complex.

    PubMed

    Crider-Pirkle, Sunday; Billingsley, Peggy; Faust, Charles; Hardy, Daniel M; Lee, Vaughan; Weitlauf, Harry

    2002-05-03

    Galectin-3 is a lectin important in animal development and regulatory processes and is found selectively localized at the implantation site of the mouse embryo. To better understand the role of galectin-3 at the maternal-fetal interface, a binding partner was isolated and characterized. Homogenates of uteroplacental tissue were incubated with immobilized recombinant galectin-3, and specifically bound proteins were eluted using lactose. The principal protein, p400, had an M(r) of 400,000 in SDS-PAGE. Physical properties of p400 and amino acid sequences of seven tryptic peptides were similar to cubilin from rats, humans, and dogs, identifying p400 as the murine ortholog of cubilin. This was further supported by the tissue distribution observed only in yolk sac, kidney, and ileum with monospecific antiserum for p400. Cubilin occurred in yolk sac epithelium throughout pregnancy, but galectin-3 was there only during the last week. Unexpectedly, cubilin was found only in perforin-containing granules of uterine natural killer (uNK) cells, although galectin-3 occurred throughout the cell cytoplasm. In situ hybridization revealed cubilin mRNA in yolk sac epithelium but not uNK cells, implying that yolk sac-derived cubilin is endocytosed by uNK cells via galectin-3. This is consistent with cubilin being an endogenous partner of galectin-3 at the maternal-fetal interface and suggests an important role for cubilin in uNK cell function.

  2. Design of a binding scaffold based on variable lymphocyte receptors of jawless vertebrates by module engineering.

    PubMed

    Lee, Sang-Chul; Park, Keunwan; Han, Jieun; Lee, Joong-jae; Kim, Hyun Jung; Hong, Seungpyo; Heu, Woosung; Kim, Yu Jung; Ha, Jae-Seok; Lee, Seung-Goo; Cheong, Hae-Kap; Jeon, Young Ho; Kim, Dongsup; Kim, Hak-Sung

    2012-02-28

    Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed "Repebody," showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

  3. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  4. In vivo aggregation properties of the nuclear poly(A)-binding protein PABPN1.

    PubMed

    Tavanez, João Paulo; Calado, Patricia; Braga, José; Lafarga, Miguel; Carmo-Fonseca, Maria

    2005-05-01

    A broad range of degenerative diseases is associated with intracellular inclusions formed by toxic, aggregation-prone mutant proteins. Intranuclear inclusions constitute a pathological hallmark of oculopharyngeal muscular dystrophy (OPMD), a dominantly inherited disease caused by (GCG) repeat expansions in the gene that encodes for nuclear poly(A) binding protein (PABPN1). The mutation results in an extended polyalanine stretch that has been proposed to induce protein aggregation and formation of intranuclear inclusions. Here we show that normal PABPN1 is inherently aggregation-prone when exogenously expressed in either HeLa or myogenic C2 cells. Similar deposits of insoluble PABPN1 are formed by variant forms of the protein containing either a polyalanine expansion or a complete deletion of the polyalanine tract, indicating that the mutation responsible for OPMD is not essential for formation of PABPN1 inclusions. In contrast, interfering with any of the protein domains required for stimulation of poly(A) polymerase prevents the formation of inclusions. Most surprisingly, photobleaching experiments reveal that both normal and expanded PABPN1 molecules are not irreversibly sequestered into aggregates, but rather move rapidly in and out of the inclusions. These findings have important implications for the interpretation of OPMD model systems based on exogenous expression of PABPN1.

  5. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

    PubMed

    Tabib-Salazar, Aline; Liu, Bing; Doughty, Philip; Lewis, Richard A; Ghosh, Somadri; Parsy, Marie-Laure; Simpson, Peter J; O'Dwyer, Kathleen; Matthews, Steve J; Paget, Mark S

    2013-06-01

    RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

  6. Palonosetron-5-HT3 Receptor Interactions As Shown by a Binding Protein Cocrystal Structure.

    PubMed

    Price, Kerry L; Lillestol, Reidun K; Ulens, Chris; Lummis, Sarah C R

    2016-12-21

    Palonosetron is a potent 5-HT3 receptor antagonist and an effective therapeutic agent against emesis. Here we identify the molecular determinants of compound recognition in the receptor binding site by obtaining a high resolution structure of palonosetron bound to an engineered acetylcholine binding protein that mimics the 5-HT3 receptor binding site, termed 5-HTBP, and by examining the potency of palonosetron in a range of 5-HT3 receptors with mutated binding site residues. The structural data indicate that palonosetron forms a tight and effective wedge in the binding pocket, made possible by its rigid tricyclic ring structure and its interactions with binding site residues; it adopts a binding pose that is distinct from the related antiemetics granisetron and tropisetron. The functional data show many residues previously shown to interact with agonists and antagonists in the binding site are important for palonosetron binding, and indicate those of particular importance are W183 (a cation-π interaction and a hydrogen bond) and Y153 (a hydrogen bond). This information, and the availability of the structure of palonosetron bound to 5-HTBP, should aid the development of novel and more efficacious drugs that act via 5-HT3 receptors.

  7. Fyn-phosphorylated PIKE-A binds and inhibits AMPK signaling, blocking its tumor suppressive activity.

    PubMed

    Zhang, S; Qi, Q; Chan, C B; Zhou, W; Chen, J; Luo, H R; Appin, C; Brat, D J; Ye, K

    2016-01-01

    The AMP-activated protein kinase, a key regulator of energy homeostasis, has a critical role in metabolic disorders and cancers. AMPK is mainly regulated by cellular AMP and phosphorylation by upstream kinases. Here, we show that PIKE-A binds to AMPK and blocks its tumor suppressive actions, which are mediated by tyrosine kinase Fyn. PIKE-A directly interacts with AMPK catalytic alpha subunit and impairs T172 phosphorylation, leading to repression of its kinase activity on the downstream targets. Mutation of Fyn phosphorylation sites on PIKE-A, depletion of Fyn, or pharmacological inhibition of Fyn blunts the association between PIKE-A and AMPK, resulting in loss of its inhibitory effect on AMPK. Cell proliferation and oncogenic assays demonstrate that PIKE-A antagonizes tumor suppressive actions of AMPK. In human glioblastoma samples, PIKE-A expression inversely correlates with the p-AMPK levels, supporting that PIKE-A negatively regulates AMPK activity in cancers. Thus, our findings provide additional layer of molecular regulation of the AMPK signaling pathway in cancer progression.

  8. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    PubMed

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells. © 2015 John Wiley & Sons Ltd.

  9. The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay

    PubMed Central

    Bresson, Stefan M.; Conrad, Nicholas K.

    2013-01-01

    Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression. PMID:24146636

  10. Acyl CoA Binding Proteins are Required for Cuticle Formation and Plant Responses to Microbes.

    PubMed

    Xia, Ye; Yu, Keshun; Gao, Qing-Ming; Wilson, Ella V; Navarre, Duroy; Kachroo, Pradeep; Kachroo, Aardra

    2012-01-01

    Fatty acids (FA) and lipids are well known regulators of plant defense. Our previous studies have shown that components of prokaryotic (plastidal) FA biosynthesis pathway regulate various aspects of plant defense. Here, we investigated the defense related roles of the soluble acyl CoA binding proteins (ACBPs), which are thought to facilitate the intracellular transport of FA/lipids. We show that ACBP3 and 4 are required for maintaining normal lipid levels and that ACBP3 contributes to the lipid flux between the prokaryotic and eukaryotic pathways. We also show that loss of ACBP3, 4, or 6 impair normal development of the cuticle and affect both basal and resistance protein-mediated defense against bacterial and fungal pathogens. Loss of ACBP3, 4, or 6 also inhibits the induction of systemic acquired resistance (SAR) due to the plants inability to generate SAR inducing signal(s). Together, these data show that ACBP3, ACBP4, and ACBP6 are required for cuticle development as well as defense against microbial pathogens.

  11. OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

    PubMed Central

    Lin, Ming-Feng; Tsai, Pei-Wen; Chen, Jeng-Yi; Lin, Yun-You; Lan, Chung-Yu

    2015-01-01

    Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding. PMID:26484669

  12. Affinity polymers tailored for the protein A binding site of immunoglobulin G proteins.

    PubMed

    Latza, Patricia; Gilles, Patrick; Schaller, Torsten; Schrader, Thomas

    2014-09-01

    Rational design in combination with a screening process was used to develop affinity polymers for a specific binding site on the surface of immunoglobulin G (IgG) proteins. The concept starts with the identification of critical amino acid residues on the protein interface and their topological arrangement. Appropriate binding monomers were subsequently synthesized. Together with a sugar monomer (2-5 equiv) for water solubility and a dansyl monomer (0.5 equiv) as a fluorescent label, they were subjected in aqueous solution to linear radical copolymerization in various compositions (e.g., azobisisobutyronitrile (AIBN), homogeneous water/DMF mixtures). After ultrafiltration and lyophilization, colorless dry water-soluble powders were obtained. NMR spectroscopic and gel permeation chromatography (GPC) characterization indicated molecular weights between 30 and 500 kD and confirmed retention of monomer composition as well as the absence of monomers. In a competitive enzyme-linked immunosorbent assay (ELISA) screen of the polymer libraries (20-50 members), few copolymers qualified as strong and selective binders for the protein A binding site on the Fc fragment of the antibody. Their monomer composition precisely reflected the critical amino acids found at the interface. The simple combination of a charged and a nonpolar binding monomer sufficed for selective submicromolar IgG recognition by the synthetic polymer. Affinities were confirmed by fluorescence titrations; they increased with decreasing salt load but remained largely unaltered at lowered pH. Other proteins, including those of similar size and isoelectric point (pI), were bound 10-1000 times less tightly. This example indicates that interaction domains in other proteins may also be targeted by synthetic polymers if their comonomer composition reflects the nature and arrangement of amino acid residues on the protein surface.

  13. IgA-binding factor suppresses synthesis of IgA in MOPC-315 plasmacytoma cells

    SciTech Connect

    Darby, C.; Moore, J.S.; Muller, S.; Aaronsen, D.; Madianos, E.; Hoover, R.G.

    1986-03-05

    T cells with Fc receptors for IgA (T/sup ..cap alpha../ cells) and their products, IgA-binding factors (IgABF), have been implicated in the regulation of IgA expression by B cells. They have previously shown that an IgABF produced by IgA induced normal T cells or constitutively by the Fc/sup ..cap alpha../ R+ T cell lymphoma, BALENTL 8, is capable of suppressing the proliferation and the amount of secreted IgA by MOPC-315 cells. In the present studies, they demonstrate that: (a) suppression of proliferation and secretion requires surface membrane IgA on the target cell, (b) suppression exhibits rapid kinetics with maximal effect occurring by 3-4 hours, (c) suppression is reversible, and (d) suppression of secretion involves selective suppression of IgA synthesis as measured by /sup 3/H-leucine incorporation into immunoprecipitable IgA(non-IgA protein is unaffected). These findings indicate that IgA-isotype-specific effector molecules interact directly with their B cell targets through surface membrane immunoglobulin and cause a down regulation of immunoglobulin synthesis by the target. Current studies are underway to address whether this selective suppression of IgA is mediated at the transcriptional, translational or post-translational level. The use of MOPC-315 tumor cells as targets of T cell produced, isotype-specific, effector molecules should provide a unique model for the further analysis of isotype regulation at the molecular level.

  14. Elevated serotonin 1A binding in remitted major depressive disorder: evidence for a trait biological abnormality.

    PubMed

    Miller, Jeffrey M; Brennan, Kathleen G; Ogden, Todd R; Oquendo, Maria A; Sullivan, Gregory M; Mann, J John; Parsey, Ramin V

    2009-09-01

    Several biological abnormalities in major depressive disorder (MDD) persist during episode remission, including altered serotonin neurotransmission, and may reflect underlying pathophysiology. We previously described elevated brain serotonin 1A (5-HT(1A)) receptor binding in antidepressant-naive (AN) subjects with MDD within a major depressive episode (MDE) compared with that in healthy controls using positron emission tomography (PET). In this study, we measured 5-HT(1A) receptor binding in unmedicated subjects with MDD during sustained remission, hypothesizing higher binding compared with that in healthy controls, and binding comparable with currently depressed AN subjects, indicative of a biological trait. We compared 5-HT(1A) binding potential (BP(F)) assessed through PET scanning with [(11)C]WAY-100635 in 15 subjects with recurrent MDD in remission for >or=12 months and off antidepressant medication for >or=6 months, 51 healthy controls, and 13 AN MDD subjects in a current MDE. Metabolite-corrected arterial input functions were acquired for the estimation of BP(F). Remitted depressed subjects had higher 5-HT(1A) BP(F) compared with healthy controls; this group difference did not vary significantly in magnitude across brain regions. 5-HT(1A) BP(F) was comparable in remitted and currently depressed subjects. Elevated 5-HT(1A) BP(F) level among subjects with remitted MDD appears to be a trait abnormality in MDD, which may underlie recurrent MDEs. Future studies should evaluate the role of genetic and environmental factors in producing elevated 5-HT(1A) BP(F) and MDD, and should examine whether 5-HT(1A) BP(F) is a vulnerability factor to MDEs that could have a role in screening high-risk populations for MDD.

  15. Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacyclic triterpenes.

    PubMed Central

    Ying, Q L; Rinehart, A R; Simon, S R; Cheronis, J C

    1991-01-01

    Several pentacyclic triterpenoid metabolites of plant origin are inhibitors of hydrolysis of both synthetic peptide substrates and elastin by human leucocyte elastase (HLE). Ursolic acid, the most potent of these compounds, has an inhibition constant of 4-6 microM for hydrolysis of peptide substrates in phosphate-buffered saline. With tripeptide and tetrapeptide substrates, the inhibition is purely competitive, whereas with a shorter dipeptide substrate the inhibition is non-competitive, suggesting that ursolic acid interacts with subsite S3 of the extended substrate-binding domain in HLE, but not with subsites S1 and S2. The carboxy group at position 28 in the pentacyclic-ring system of the triterpenes contributes to binding to HLE, since replacement of this group with a hydroxy group, as in uvaol, the alcohol analogue of ursolic acid, reduces the potency of inhibition. The inhibitory potency of ursolic acid is also reduced by addition of 1 M-NaCl, further supporting a postulated electrostatic interaction between the negative charge on the triterpene and a positively charged residue on the enzyme, which we assign to the side chain of Arg-217, located in the vicinity of subsites S4 and S5 in HLE. These observations are consistent with a binding site for ursolic acid which extends from S3 towards S4 and S5 on the enzyme. Other triterpenes, including oleanolic acid, erythrodiol, hederagenin and 18 beta-glycyrrhetic acid, can also interact with this binding site. On the basis of these results we conclude that the extended substrate-binding domain of HLE can accommodate a variety of hydrophobic ligands, including not only such molecules as fatty acids [Ashe & Zimmerman (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook & Ternai (1988) Biol. Chem. Hoppe-Seyler 369, 629-637], but also polycyclic molecules such as the pentacyclic triterpenoids. PMID:1859379

  16. Poly (A) Binding Protein Cytoplasmic 1 Is a Novel Co-Regulator of the Androgen Receptor

    PubMed Central

    Eisermann, Kurtis; Dar, Javid A.; Dong, Jun; Wang, Dan; Masoodi, Khalid Z.; Wang, Zhou

    2015-01-01

    The androgen receptor (AR) is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner. The NTD of the AR plays a key role in AR transactivation including androgen-independent activation of the AR in castration-resistant prostate cancer (CRPC) cells. We recently reported that amino acids (a.a.) 50-250 of the NTD are capable of modulating AR nucleocytoplasmic trafficking. To further explore the mechanism associated with a.a. 50-250, GFP pull-down assays were performed in C4-2 CRPC cells transfected with GFP tagged a.a. 50-250 of the AR. Mass spectrometry analysis of the pulled down proteins identified poly (A) binding protein cytoplasmic 1 (PABPC1) interaction with this region of the AR. In silico analysis of gene expression data revealed PABPC1 up-regulation in prostate cancer tissue specimens and this up-regulation correlates to increased disease recurrence. Co-immunoprecipitation assays confirmed the association of PABPC1 with a.a. 50-250 of the NTD of the AR. Knockdown of PABPC1 decreased nuclear AR protein levels and inhibited androgen activation of the AR target PSA in LNCaP and C4-2 cells. Additionally, knockdown of PABPC1 inhibited transactivation of the PSA promoter by NAR (AR lacking the LBD) and attenuated proliferation of AR-positive prostate cancer cells. These findings suggest that PABPC1 is a novel co-regulator of the AR and may be a potential target for blocking activation of the AR in CRPC. PMID:26176602

  17. PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor

    PubMed Central

    Bucur, Octavian; Stancu, Andreea Lucia; Muraru, Maria Sinziana; Melet, Armelle; Petrescu, Stefana Maria; Khosravi-Far, Roya

    2015-01-01

    FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ’s development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor. PMID:26280018

  18. PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor.

    PubMed

    Bucur, Octavian; Stancu, Andreea Lucia; Muraru, Maria Sinziana; Melet, Armelle; Petrescu, Stefana Maria; Khosravi-Far, Roya

    2014-01-01

    FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ's development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor.

  19. Molecular properties of the class III subfamily of acyl-coenyzme A binding proteins from tung tree (Vernicia fordii)

    USDA-ARS?s Scientific Manuscript database

    Acyl-CoA binding proteins (ACBPs) have been identified in most branches of life. A single prototypical ACBP was first discovered in yeast, and was found to play a signficant role in lipid metabolism, among other functions. Plants also contain the prototype small, soluble ACBP, but have also evolve...

  20. Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages.

    PubMed

    Setiawan, Laurentia C; van Dort, Karel A; Rits, Maarten A N; Kootstra, Neeltje A

    2016-04-01

    Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.

  1. Brain monoamine oxidase A binding in major depressive disorder: relationship to selective serotonin reuptake inhibitor treatment, recovery, and recurrence.

    PubMed

    Meyer, Jeffrey H; Wilson, Alan A; Sagrati, Sandra; Miler, Laura; Rusjan, Pablo; Bloomfield, Peter M; Clark, Michael; Sacher, Julia; Voineskos, Aristotle N; Houle, Sylvain

    2009-12-01

    Highly significant elevations in regional brain monoamine oxidase A (MAO-A) binding were recently reported during major depressive episodes (MDEs) of major depressive disorder (MDD). The relationship between MAO-A levels and selective serotonin reuptake inhibitor (SSRI) treatment, recovery, and recurrence in MDD is unknown. To determine whether brain MAO-A binding changes after SSRI treatment, whether brain MAO-A binding normalizes in subjects with MDD in recovery, and whether there is a relationship between prefrontal and anterior cingulate cortex MAO-A binding in recovery and subsequent recurrence of MDE. Case-control study. Tertiary care psychiatric hospital. Twenty-eight healthy subjects, 16 subjects with an MDE secondary to MDD, and 18 subjects with MDD in recovery underwent carbon 11-labeled harmine positron emission tomography scans. Subjects with MDE were scanned before and after 6 weeks of SSRI treatment. All were otherwise healthy, nonsmoking, and medication free. Subjects with MDD in recovery were followed up for 6 months after MAO-A binding measurement. Monoamine oxidase A V(T), an index of MAO-A density, was measured in the prefrontal cortex, anterior cingulate cortex, posterior cingulate cortex, dorsal putamen, ventral striatum, thalamus, anterior temporal cortex, midbrain, and hippocampus. Monoamine oxidase A V(T) was significantly elevated in each brain region both during MDE and after SSRI treatment as compared with healthy controls. During recovery, MAO-A V(T) was significantly elevated in each brain region; however, those who went on to recurrence had significantly higher MAO-A V(T) in the prefrontal and anterior cingulate cortex than those who did not. Elevated MAO-A binding after SSRI treatment indicates persistence of a monoamine-lowering process not present in health. This provides a strong conceptual rationale for continuing SSRI treatment during early remission. Greater MAO-A binding in the prefrontal and anterior cingulate cortex in

  2. Phylogenetic analysis reveals dynamic evolution of the poly(A)-binding protein gene family in plants.

    PubMed

    Gallie, Daniel R; Liu, Renyi

    2014-11-25

    The poly(A)-binding protein (PABP) binds the poly(A) tail of eukaryotic mRNAs and functions to maintain the integrity of the mRNA while promoting protein synthesis through its interaction with eukaryotic translation initiation factor (eIF) 4G and eIF4B. PABP is encoded by a single gene in yeast and marine algae but during plant evolution the PABP gene family expanded substantially, underwent sequence divergence into three subclasses, and acquired tissue-specificity in gene family member expression. Although such changes suggest functional specialization, the size of the family and its sequence divergence have complicated an understanding of which gene family members may be foundational and which may represent more recent expansions of the family to meet the specific needs of speciation. Here, we examine the evolution of the plant PABP gene family to provide insight into these aspects of the family that may yield clues into the function of individual family members. The PABP gene family had expanded to two members by the appearance of fresh water algae and four members in non-vascular plants. In lycophytes, the first sequence divergence yielding a specific class member occurs. The earliest members of the gene family share greatest similarity to those modern members whose expression is confined to reproductive tissues, suggesting that supporting reproductive-associated gene expression is the most conserved function of this family. A family member sharing similarity to modern vegetative-associated members first appears in gymnosperms. Further elaboration of the reproductive-associated and vegetative-associated members occurred during the evolution of flowering plants. Expansion of the plant PABP gene family began prior to the colonization of land. By the evolution of lycophytes, the first class member whose expression is confined to reproductive tissues in higher plants had appeared. A second class member whose expression is vegetative-associated appeared in

  3. Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

    PubMed

    Woods, Alison J; Roberts, Marnie S; Choudhary, Jyoti; Barry, Simon T; Mazaki, Yuichi; Sabe, Hisataka; Morley, Simon J; Critchley, David R; Norman, Jim C

    2002-02-22

    Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

  4. Monoamine oxidase A binding in the prefrontal and anterior cingulate cortices during acute withdrawal from heavy cigarette smoking.

    PubMed

    Bacher, Ingrid; Houle, Sylvain; Xu, Xin; Zawertailo, Laurie; Soliman, Alexandra; Wilson, Alan A; Selby, Peter; George, Tony P; Sacher, Julia; Miler, Laura; Kish, Stephen J; Rusjan, Pablo; Meyer, Jeffrey H

    2011-08-01

    Greater prefrontal cortex and anterior cingulate cortex monoamine oxidase A (MAO-A) binding is associated with depressed mood. Substances in cigarette smoke, such as harman, inhibit MAO-A, and cigarette withdrawal is associated with depressed mood. Dysphoria during cigarette withdrawal predicts relapse. It is unknown whether MAO-A binding increases during early cigarette withdrawal. To measure prefrontal and anterior cingulate cortex MAO-A binding during acute cigarette withdrawal and to assess the relationship with smoking severity, plasma levels of harman, and severity of depression. Study via positron emission tomography of healthy control and cigarette-smoking individuals. Twenty-four healthy nonsmoking and 24 otherwise healthy cigarette-smoking individuals underwent positron emission tomography with harmine labeled with carbon 11. Healthy nonsmoking individuals underwent scanning once. Cigarette-smoking individuals underwent scanning after acute withdrawal and after active cigarette smoking. Cigarette smoking was heavy (≥25 cigarettes per day) or moderate (15-24 cigarettes per day). Tertiary care psychiatric hospital. An index of MAO-A density, MAO-A V(T), was measured in the prefrontal and anterior cingulate cortices. In heavy-smoking individuals, prefrontal and anterior cingulate cortex MAO-A V(T) was greater during withdrawal (23.7% and 33.3%, respectively; repeated-measures multivariate analysis of variance, F(1,22) = 25.58, P < .001). During withdrawal from heavy smoking, prefrontal and anterior cingulate cortex MAO-A V(T) was greater than in healthy controls (25.0% and 25.6%, respectively; multivariate analysis of variance, F(2,33) = 6.72, P = .004). The difference in MAO-A V(T) in the prefrontal cortex and anterior cingulate cortex between withdrawal and active, heavy smoking covaried with change in plasma harman levels in the prefrontal cortex and anterior cingulate cortex (multivariate analysis of covariance, F(1,10) = 9.97, P = .01). The change in

  5. The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila

    PubMed Central

    Liew, Gerald M.; Foulk, Michael S.

    2014-01-01

    The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate themolecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification. PMID:23737076

  6. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    NASA Astrophysics Data System (ADS)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  7. RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations.

    PubMed

    Leger, J F; Robert, J; Bourdieu, L; Chatenay, D; Marko, J F

    1998-10-13

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

  8. RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

    PubMed Central

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-01-01

    Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions. PMID:9770480

  9. Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation.

    PubMed

    Dai, Lixin; Taylor, Martin S; O'Donnell, Kathryn A; Boeke, Jef D

    2012-11-01

    Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell.

  10. Negative regulation of meiotic gene expression by the nuclear poly(a)-binding protein in fission yeast.

    PubMed

    St-André, Olivier; Lemieux, Caroline; Perreault, Audrey; Lackner, Daniel H; Bähler, Jürg; Bachand, François

    2010-09-03

    Meiosis is a cellular differentiation process in which hundreds of genes are temporally induced. Because the expression of meiotic genes during mitosis is detrimental to proliferation, meiotic genes must be negatively regulated in the mitotic cell cycle. Yet, little is known about mechanisms used by mitotic cells to repress meiosis-specific genes. Here we show that the poly(A)-binding protein Pab2, the fission yeast homolog of mammalian PABPN1, controls the expression of several meiotic transcripts during mitotic division. Our results from chromatin immunoprecipitation and promoter-swapping experiments indicate that Pab2 controls meiotic genes post-transcriptionally. Consistently, we show that the nuclear exosome complex cooperates with Pab2 in the negative regulation of meiotic genes. We also found that Pab2 plays a role in the RNA decay pathway orchestrated by Mmi1, a previously described factor that functions in the post-transcriptional elimination of meiotic transcripts. Our results support a model in which Mmi1 selectively targets meiotic transcripts for degradation via Pab2 and the exosome. Our findings have therefore uncovered a mode of gene regulation whereby a poly(A)-binding protein promotes RNA degradation in the nucleus to prevent untimely expression.

  11. Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-05-13

    Highlights: {yields} Depletion of cellular PABP level arrests mRNA translation in HeLa cells. {yields} PABP knock down leads to apoptotic cell death. {yields} PABP depletion does not affect transcription. {yields} PABP depletion does not lead to nuclear accumulation of mRNA. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

  12. Functional compensation for the loss of testis-specific poly(A)-binding protein, PABPC2, during mouse spermatogenesis

    PubMed Central

    KASHIWABARA, Shin-ichi; TSURUTA, Satsuki; OKADA, Keitaro; SAEGUSA, Ayaka; MIYAGAKI, Yu; BABA, Tadashi

    2016-01-01

    Mouse testes contain several isoforms of cytoplasmic poly(A)-binding proteins (PABPCs), including ubiquitous PABPC1 and testis-specific PABPC2/PABPt. PABPC2 is characterized by its absence from translationally active polyribosomes and elongating spermatids. To elucidate the function of PABPC2 in spermatogenesis, we produced mutant mice lacking PABPC2. The PABPC2-null mice showed normal fertility. The processes of spermatogenesis and sperm migration in the testes and epididymides, respectively, were normal in the mutant mice. When the involvement of PABPC2 in translational regulation of haploid-specific mRNAs was examined, these mRNAs were correctly transcribed in round spermatids and translated in elongating spermatids. Moreover, immunoblot analysis revealed low abundance of PABPC2 relative to PABPC1 in spermatogenic cells. These results suggest that PABPC2 may be either functionally redundant with other PABPCs (including PABPC1) or largely dispensable for translational regulation during spermiogenesis. PMID:26971890

  13. Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

    PubMed

    Bansal, Dhiru; Kulkarni, Jahnavi; Nadahalli, Kavana; Lakshmanan, Vairavan; Krishna, Srikar; Sasidharan, Vidyanand; Geo, Jini; Dilipkumar, Shilpa; Pasricha, Renu; Gulyani, Akash; Raghavan, Srikala; Palakodeti, Dasaradhi

    2017-09-01

    Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration. © 2017. Published by The Company of Biologists Ltd.

  14. Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

    PubMed Central

    Wormington, M; Searfoss, A M; Hurney, C A

    1996-01-01

    The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability. Images PMID:8631310

  15. Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice

    PubMed Central

    Yang, Cai-Rong; Lowther, Katie M.; Lalioti, Maria D.

    2016-01-01

    Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab−/− female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab−/− mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab−/− oocytes, or when Epab−/− OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab−/− mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab−/− CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab−/− oocytes express these proteins at comparable levels with WT. Epab−/− granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function. PMID:26492470

  16. Embryonic poly(A)-binding protein (EPAB) is required for oocyte maturation and female fertility in mice

    PubMed Central

    Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Aydiner, Fulya; Sasson, Isaac; Ilbay, Orkan; Sakkas, Denny; Lowther, Katie M.; Mehlmann, Lisa M.; Seli, Emre

    2014-01-01

    Gene expression during oocyte maturation and early embryogenesis up to zygotic genome activation requires translational activation of maternally-derived mRNAs. EPAB [embryonic poly(A)-binding protein] is the predominant poly(A)-binding protein during this period in Xenopus, mouse and human. In Xenopus oocytes, ePAB stabilizes maternal mRNAs and promotes their translation. To assess the role of EPAB in mammalian reproduction, we generated Epab-knockout mice. Although Epab−/− males and Epab+/− of both sexes were fertile, Epab−/− female mice were infertile, and could not generate embryos or mature oocytes in vivo or in vitro. Epab−/− oocytes failed to achieve translational activation of maternally-stored mRNAs upon stimulation of oocyte maturation, including Ccnb1 (cyclin B1) and Dazl (deleted in azoospermia-like) mRNAs. Microinjection of Epab mRNA into Epab−/− germinal vesicle stage oocytes did not rescue maturation, suggesting that EPAB is also required for earlier stages of oogenesis. In addition, late antral follicles in the ovaries of Epab−/− mice exhibited impaired cumulus expansion, and a 8-fold decrease in ovulation, associated with a significant down-regulation of mRNAs encoding the EGF (epidermal growth factor)-like growth factors Areg (amphiregulin), Ereg (epiregulin) and Btc (betacellulin), and their downstream regulators, Ptgs2 (prostaglandin synthase 2), Has2 (hyaluronan synthase 2) and Tnfaip6 (tumour necrosis factor α-induced protein 6). The findings from the present study indicate that EPAB is necessary for oogenesis, folliculogenesis and female fertility in mice. PMID:22621333

  17. Embryonic Poly(A)-Binding Protein (EPAB) Is Required for Granulosa Cell EGF Signaling and Cumulus Expansion in Female Mice.

    PubMed

    Yang, Cai-Rong; Lowther, Katie M; Lalioti, Maria D; Seli, Emre

    2016-01-01

    Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos before zygotic genome activation. EPAB is required for translational activation of maternally stored mRNAs in the oocyte and Epab(-/-) female mice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aim of this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab(-/-) mice. Using a coculture system of oocytectomized cumulus oophorus complexes (OOXs) with denuded oocytes, we found that when wild-type OOXs were cocultured with Epab(-/-) oocytes, or when Epab(-/-) OOXs were cocultured with WT oocytes, cumulus expansion failed to occur in response to epidermal growth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab(-/-) mice fail to send and receive the necessary signals required for cumulus expansion. The abnormalities in Epab(-/-) CCs are not due to lower expression of the oocyte-derived factors growth differentiation factor 9 or bone morphogenetic protein 15, because Epab(-/-) oocytes express these proteins at comparable levels with WT. Epab(-/-) granulosa cells (GCs) exhibit decreased levels of phosphorylated MEK1/2, ERK1/2, and p90 ribosomal S6 kinase in response to lutenizing hormone and EGF treatment, as well as decreased phosphorylation of the EGF receptor. In conclusion, EPAB, which is oocyte specific, is required for the ability of CCs and GCs to become responsive to LH and EGF signaling. These results emphasize the importance of oocyte-somatic communication for GC and CC function.

  18. Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis.

    PubMed

    Kasho, Kazutoshi; Tanaka, Hiroyuki; Sakai, Ryuji; Katayama, Tsutomu

    2017-01-27

    Timely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaA-bound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF·DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF·DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.

  19. Examination of the transcription factor NtcA-binding motif by in vitro selection of DNA sequences from a random library.

    PubMed

    Jiang, F; Wisén, S; Widersten, M; Bergman, B; Mannervik, B

    2000-08-25

    A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N(8))-TACA, was found to be the optimal NtcA-binding sequence. The more divergent the binding sequences, compared to this consensus sequence, the lower the NtcA affinity. The second and third bases in each four-nucleotide half of the consensus sequence were crucial for NtcA binding, and they were in general highly conserved. The most frequently occurring sequence in the middle weakly conserved region was similar to that of the NtcA-binding motif of the Anabaena sp. strain PCC 7120 glnA gene, previously known to have high affinity for NtcA. This indicates that the middle sequences were selected for high NtcA affinity. Analysis of natural NtcA-binding motifs showed that these could be classified into two groups based on differences in recognition consensus sequences. It is suggested that NtcA naturally recognizes different DNA-binding motifs, or has differential affinities to these sequences under different physiological conditions.

  20. Nuclear AP/sub 4/A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

    SciTech Connect

    Morioka, M.; Shimada, H.

    1986-01-01

    The AP/sub 4/A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5', 5'''-P/sup 1/,P/sup 4/-tetraphosphate (Ap/sub 4/A). Among various subcellular components that can bind (/sup 3/H)AP/sub 4/A, nuclei alone showed the highly specific Ap/sub 4/A-binding activity which was not influenced by the presence of AP/sub 4/A, AP/sub 5/A and GP/sub 4/G. The addition of an excess amount of ATP only slightly reduced the binding of (/sup 3/H)AP/sub 4/A to the nuclei. It was found that AP/sub 4/A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP/sub 4/A-binding activity fluctuated cyclically during each cell cycle, with at transient increase at the beginning of S phase followed by an abrupt-decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP/sub 4/A-binding activity was also prevented. It seems that the binding of AP/sub 4/A to the nuclear structural protein is involved in the initiation of S phase.

  1. Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking.

    PubMed

    Leung, Yuk M; Kang, Youhou; Gao, Xiaodong; Xia, Fuzhen; Xie, Huanli; Sheu, Laura; Tsuk, Sharon; Lotan, Ilana; Tsushima, Robert G; Gaisano, Herbert Y

    2003-05-09

    Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.

  2. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    PubMed

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered.

  3. Drosophila Larp associates with poly(A)-binding protein and is required for male fertility and syncytial embryo development.

    PubMed

    Blagden, Sarah P; Gatt, Melanie K; Archambault, Vincent; Lada, Karolina; Ichihara, Keiko; Lilley, Kathryn S; Inoue, Yoshihiro H; Glover, David M

    2009-10-01

    As the influence of mRNA translation upon cell cycle regulation becomes clearer, we searched for genes that might specify such control in Drosophila. A maternal-effect lethal screen identified mutants in the Drosophila gene for Larp (La-related protein) which displayed maternal-effect lethality and male sterility. A role for La protein has already been implicated in mRNA translation whereas Larp has been proposed to regulate mRNA stability. Here we demonstrate that Larp exists in a physical complex with, and also interacts genetically with, the translation regulator poly(A)-binding protein (PABP). Most mutant alleles of pAbp are embryonic lethal. However hypomorphic pAbp alleles show similar meiotic defects to larp mutants. We find that larp mutant-derived syncytial embryos show a range of mitotic phenotypes, including failure of centrosomes to migrate around the nuclear envelope, detachment of centrosomes from spindle poles, the formation of multipolar spindle arrays and cytokinetic defects. We discuss why the syncytial mitotic cycles and male meiosis should have a particularly sensitive requirement for Larp proteins in regulating not only transcript stability but also potentially the translation of mRNAs.

  4. Arginyl residues are involved in acyl-CoA binding to the elongase from etiolated leek seedlings.

    PubMed

    Santarelli, X; Chevalier, S; Cassagne, C; Lessire, R

    1998-04-22

    The C18:0-CoA elongase from etiolated leek seedling microsomes was inactivated by treatment with phenylglyoxal, a reagent which specifically modifies arginyl residues. In the presence of 20 mM phenylglyoxal, 95% of the C18:0-CoA elongation was inhibited. The condensation and dehydration reactions of the overall elongation were totally inhibited, whereas enoyl-CoA reductase activity was diminished by 75%, but the nature of the final elongation product was unchanged. Phenylglyoxal did not modify the C18:0-CoA partition between membrane and aqueous compartments; moreover, [1-14C]phenylglyoxal labeling experiments showed a covalent binding of the inhibitor to membrane proteins. The ability of several substrates to prevent the inactivation by phenylglyoxal was investigated. NADH and NADPH had no effect. CoA led to a 75% protection, and the incorporation of [14C]phenylglyoxal was strongly affected by 10 mM CoA. The acyl chain length of the acyl-CoAs played also a crucial role in preventing the binding of phenylglyoxal. The maximal prevention of phenylglyoxal inhibition was obtained with C18:0-CoA. This suggests that arginyl residues could be present in the vicinity of the acyl-CoA binding site of the subunits of C18:0-CoA elongase. Copyright 1998 Elsevier Science B.V.

  5. The nuclear poly(A) binding protein of mammals, but not of fission yeast, participates in mRNA polyadenylation.

    PubMed

    Kühn, Uwe; Buschmann, Juliane; Wahle, Elmar

    2017-04-01

    The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of ∼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors.

  6. Partial purification of a binding protein for polychlorinated biphenyls from rat lung cytosol: Physicochemical and immunochemical characterization

    SciTech Connect

    Lund, J.; Nordlund, L.; Gustafsson, J.A. )

    1988-10-04

    A binding protein for certain methyl sulfone metabolites of polychlorinated biphenyls (PCB) was partially purified from lung cytosol of untreated female rats. The protein has an M{sub r} of 13,000 and a pI of 5.3 in the absence of reducing agents. In the presence of dithioerythreitol or {beta}-mercaptoethanol, the protein is split into subunits with a more basic pI. The 13-kDa protein was electroeluted from SDS-polyacrylamide gels, and an antiserum against the protein was raised in rabbit. The immunoglobulin fraction was shown to contain monospecific antibodies against the 13-kDa protein as determined by Western immunoblots. Due to striking similarities in physicochemical characteristics of the 13-kDa protein and a protein purified from rabbit lung and uterus, uteroglobin, the anti 13-kDa protein antibodies were tested for cross-reactivity. As judged by Western immunoblots, the anti 13-kDa protein antibodies did not cross-react with uteroglobin and the two proteins, although similar, do not seem to be identical. The 13-kDa protein is proposed to be responsible for the accumulation of certain methylsulfonyl-PCBs in lung tissue of rats. Monospecific antibodies against the 13-kDa protein should constitute immunochemical probes of great value in attempts to elucidate the physiological role of the protein as well as its possible role in PCB-induced respiratory toxicity.

  7. The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs.

    PubMed

    Lemay, Jean-François; D'Amours, Annie; Lemieux, Caroline; Lackner, Daniel H; St-Sauveur, Valérie G; Bähler, Jürg; Bachand, François

    2010-01-15

    Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs. Copyright 2010 Elsevier Inc. All rights reserved.

  8. SP-A binds alpha1-antitrypsin in vitro and reduces the association rate constant for neutrophil elastase

    PubMed Central

    Gorrini, Marina; Lupi, Anna; Iadarola, Paolo; Santos, Conceição Dos; Rognoni, Paola; Dalzoppo, Daniele; Carrabino, Natalia; Pozzi, Ernesto; Baritussio, Aldo; Luisetti, Maurizio

    2005-01-01

    Background α1-antitrypsin and surfactant protein-A (SP-A) are major lung defense proteins. With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of in vitro experiments aimed at investigating the nature and consequences of such an interaction. Methods and results At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin. Conclusion We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents. PMID:16351724

  9. Bacillus anthracis S-layer protein BslA binds to extracellular matrix by interacting with laminin.

    PubMed

    Wang, Yanchun; Wei, Ying; Yuan, Shengling; Tao, Haoxia; Dong, Jie; Zhang, Zhaoshan; Tian, Wei; Liu, Chunjie

    2016-08-11

    The Bacillus anthracis S-layer protein, BslA, plays a crucial role in mammalian infection. BslA is required to mediate adherence between host cells and vegetative forms of bacteria and this interaction promotes target organs adherence and blood-brain barrier (BBB) penetration in vivo. This study attempts to identify the potential eukaryotic ligand(s) for B. anthracis BslA protein. Biochemical approaches have indicated that the putative host cell ligand(s) for BslA is a surface protein, which is independent of the sugar components for binding to Bs1A. A ligand screening using blot overlays, far Western blots and mass spectrometry analyses revealed that BslA binds to mammalian laminin. ELISA based solid-phase binding assays and surface plasmon resonance assays demonstrated that there were high affinity interactions between BslA(260-652) and laminin. The SPR results also revealed the dissociation constants values of 3.172 × 10(-9)M for the binding of BslA(260-652) to laminin. These data demonstrated that laminin is a ligand for BslA.

  10. The acyl-CoA binding protein is required for normal epidermal barrier function in mice[S

    PubMed Central

    Bloksgaard, Maria; Bek, Signe; Marcher, Ann-Britt; Neess, Ditte; Brewer, Jonathan; Hannibal-Bach, Hans Kristian; Helledie, Torben; Fenger, Christina; Due, Marianne; Berzina, Zane; Neubert, Reinhard; Chemnitz, John; Finsen, Bente; Clemmensen, Anders; Wilbertz, Johannes; Saxtorph, Henrik; Knudsen, Jens; Bagatolli, Luis; Mandrup, Susanne

    2012-01-01

    The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP−/− mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP−/− mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP+/+ and ACBP−/− mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP−/− mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP−/− mice display a ∼50% increased transepidermal water loss compared with ACBP+/+ mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function. PMID:22829653

  11. Acyl-CoA-binding domain containing 3 modulates NAD+ metabolism through activating poly(ADP-ribose) polymerase 1.

    PubMed

    Chen, Yong; Bang, Sookhee; Park, Soohyun; Shi, Hanyuan; Kim, Sangwon F

    2015-07-15

    NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function.

  12. Specific regulation of low-abundance transcript variants encoding human Acyl-CoA binding protein (ACBP) isoforms

    PubMed Central

    Nitz, Inke; Kruse, Marie-Luise; Klapper, Maja; Döring, Frank

    2011-01-01

    Abstract Despite intensive efforts on annotation of eukaryotic transcriptoms, little is known about the regulation of low-abundance transcripts. To address this question, we analysed the regulation of novel low-abundance transcript variants of human acyl-CoA binding protein (ACBP), an important multifunctional housekeeping protein, which we have identified by screening of human expressed sequence tags in combination with ab initio gene prediction. By using RT-, real-time RT- and rapid amplification of cDNA ends-PCR in five human tissues, we find these transcripts, which are generated by a consequent use of alternative promoters and alternate first or first two exons, to be authentic ones. They show a tissue-specific distribution and intrinsic responsiveness to glucose and insulin. Promoter analyses of the corresponding transcripts revealed a differential regulation mediated by sterol regulatory element-binding protein-2, hepatocyte nuclear factor-4α and nuclear factor κB (NF-κB), central transcription factors of fat and glucose metabolism and inflammation. Subcellular localization studies of deduced isoforms in liver HepG2 cells showed that they are distributed in different compartments. By demonstrating that ACBP is a target of NF-κB, our findings link fatty acid metabolism with inflammation. Furthermore, our findings show that low-abundance transcripts are regulated in a similar mode than their high-abundance counterparts. PMID:20345851

  13. Investigating the Turing conditions for diffusion-driven instability in the presence of a binding immobile substrate.

    PubMed

    Korvasová, K; Gaffney, E A; Maini, P K; Ferreira, M A; Klika, V

    2015-02-21

    Turing's diffusion-driven instability for the standard two species reaction-diffusion system is only achievable under well-known and rather restrictive conditions on both the diffusion rates and the kinetic parameters, which necessitates the pairing of a self-activator with a self-inhibitor. In this study we generalize the standard two-species model by considering the case where the reactants can bind to an immobile substrate, for instance extra-cellular matrix, and investigate the influence of this dynamics on Turing's diffusion-driven instability. Such systems have been previously studied on the grounds that binding of the self-activator to a substrate may effectively reduce its diffusion rate and thus induce a Turing instability for species with equal diffusion coefficients, as originally demonstrated by Lengyel and Epstein (1992) under the assumption that the bound state dynamics occurs on a fast timescale. We, however, analyse the full system without any separation of timescales and demonstrate that the full system also allows a relaxation of the standard constraints on the reaction kinetics for the Turing instability, increasing the type of interactions that could give rise to spatial patterning. In particular, we show that two self-activators can undertake a diffusively driven instability in the presence of a binding immobile substrate, highlighting that the interactions required of a putative biological Turing instability need not be associated with a self-activator-self-inhibitor morphogen pair.

  14. The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress tolerant phenotypes

    PubMed Central

    Martani, Francesca; Marano, Francesca; Bertacchi, Stefano; Porro, Danilo; Branduardi, Paola

    2015-01-01

    When exploited as cell factories, Saccharomyces cerevisiae cells are exposed to harsh environmental stresses impairing titer, yield and productivity of the fermentative processes. The development of robust strains therefore represents a pivotal challenge for the implementation of cost-effective bioprocesses. Altering master regulators of general cellular rewiring represents a possible strategy to evoke shaded potential that may accomplish the desirable features. The poly(A) binding protein Pab1, as stress granules component, was here selected as the target for obtaining widespread alterations in mRNA metabolism, resulting in stress tolerant phenotypes. Firstly, we demonstrated that the modulation of Pab1 levels improves robustness against different stressors. Secondly, the mutagenesis of PAB1 and the application of a specific screening protocol on acetic acid enriched medium allowed the isolation of the further ameliorated mutant pab1 A60-9. These findings pave the way for a novel approach to unlock industrially promising phenotypes through the modulation of a post-transcriptional regulatory element. PMID:26658950

  15. Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains.

    PubMed

    Dick, Gunnar; Tan, Chin Lik; Alves, Joao Nuno; Ehlert, Erich M E; Miller, Gregory M; Hsieh-Wilson, Linda C; Sugahara, Kazuyuki; Oosterhof, Arie; van Kuppevelt, Toin H; Verhaagen, Joost; Fawcett, James W; Kwok, Jessica C F

    2013-09-20

    Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186-200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.

  16. Carbonation as a binding mechanism for coal/calcium hydroxide pellets. Final technical report, September 1, 1991--August 31, 1992

    SciTech Connect

    Rapp, D.M.; Lytle, J.M.; Hackley, K.C.; Strickland, R.; Berger, R.; Schanche, G.

    1992-12-31

    In this project, the ISGS is investigating the pelletization of fine coal with calcium hydroxide, a sulfur-capturing sorbent. The objective is to produce a readily-transportable fuel which will burn in compliance with the recently passed Clean Air Act Amendment (CAAA). To improve the economics of pelletizing, carbonation, or, the reaction of carbon dioxide with calcium hydroxide, which produces a binding matrix of calcium carbonate, is being investigated as a method of hardening pelletized coal fines. This year, pellets were produced from 28 {times} 0 coal fines collected from an Illinois preparation plant using a laboratory version of a California Pellet Mill (CPM), a commercially available pellet machine. The CPM effectively pelletized coal fines at the moisture content they were dewatered to at the plant. Carbonation nearly doubled the strength of pellets containing 10 wt % calcium hydroxide. Other results from this year`s work indicate that inclusion of calcium hydroxide into pellets resulted in chlorine capture of approximately 20 wt % for combustion tests conducted at both 850 and 1100{degrees}C. Arsenic emissions were reduced from near 38 wt% at 850 C to essentially nil with inclusion of 10 wt % calcium hydroxide into the pellets. At 110{degrees}C, arsenic emissions were reduced from about 90 wt % to about 15 wt %. Sodium emissions, however, increased with the addition of calcium hydroxide. At 850{degrees}C, sodium capture dropped from about 98 wt % to 73 wt % for pellets containing 10 wt % calcium hydroxide; at 1100{degrees}C, capture dropped from about 92 wt % to about 20 wt %.

  17. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

    PubMed Central

    Zhang, Chongxu; Nielsen, Maria E. O.; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J.; Andersen, Jens S.; Yao, Gang

    2013-01-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1’s defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made. PMID:22836166

  18. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

    PubMed

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

    2012-09-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

  19. Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains*

    PubMed Central

    Dick, Gunnar; Tan, Chin Lik; Alves, Joao Nuno; Ehlert, Erich M. E.; Miller, Gregory M.; Hsieh-Wilson, Linda C.; Sugahara, Kazuyuki; Oosterhof, Arie; van Kuppevelt, Toin H.; Verhaagen, Joost; Fawcett, James W.; Kwok, Jessica C. F.

    2013-01-01

    Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186–200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity. PMID:23940048

  20. Polyadenylation-Dependent Control of Long Noncoding RNA Expression by the Poly(A)-Binding Protein Nuclear 1

    PubMed Central

    Beaulieu, Yves B.; Kleinman, Claudia L.; Landry-Voyer, Anne-Marie; Majewski, Jacek; Bachand, François

    2012-01-01

    The poly(A)-binding protein nuclear 1 (PABPN1) is a ubiquitously expressed protein that is thought to function during mRNA poly(A) tail synthesis in the nucleus. Despite the predicted role of PABPN1 in mRNA polyadenylation, little is known about the impact of PABPN1 deficiency on human gene expression. Specifically, it remains unclear whether PABPN1 is required for general mRNA expression or for the regulation of specific transcripts. Using RNA sequencing (RNA–seq), we show here that the large majority of protein-coding genes express normal levels of mRNA in PABPN1–deficient cells, arguing that PABPN1 may not be required for the bulk of mRNA expression. Unexpectedly, and contrary to the view that PABPN1 functions exclusively at protein-coding genes, we identified a class of PABPN1–sensitive long noncoding RNAs (lncRNAs), the majority of which accumulated in conditions of PABPN1 deficiency. Using the spliced transcript produced from a snoRNA host gene as a model lncRNA, we show that PABPN1 promotes lncRNA turnover via a polyadenylation-dependent mechanism. PABPN1–sensitive lncRNAs are targeted by the exosome and the RNA helicase MTR4/SKIV2L2; yet, the polyadenylation activity of TRF4-2, a putative human TRAMP subunit, appears to be dispensable for PABPN1–dependent regulation. In addition to identifying a novel function for PABPN1 in lncRNA turnover, our results provide new insights into the post-transcriptional regulation of human lncRNAs. PMID:23166521

  1. FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets

    PubMed Central

    Begonja, Antonija Jurak; Pluthero, Fred G.; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W.; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M.; Kahr, Walter H. A.; Hartwig, John H.

    2015-01-01

    Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. PMID:25838348

  2. Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase*

    PubMed Central

    Demmer, Ulrike; Warkentin, Eberhard; Srivastava, Ankita; Kockelkorn, Daniel; Pötter, Markus; Marx, Achim; Fuchs, Georg; Ermler, Ulrich

    2013-01-01

    Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis. PMID:23325803

  3. Molecular geometry of CsrA (RsmA) binding to RNA and its implications for regulated expression.

    PubMed

    Mercante, Jeffrey; Edwards, Adrianne N; Dubey, Ashok K; Babitzke, Paul; Romeo, Tony

    2009-09-18

    The global regulatory protein CsrA binds to the 5'-untranslated leader of target transcripts and alters their translation and/or stability. CsrA is a symmetrical homodimer containing two identical RNA-binding surfaces. Gel shift assays with model RNA substrates now show that CsrA can bind simultaneously at two target sites within a transcript (bridging or dual-site binding). An intersite distance of approximately 18 nucleotides (nt) was optimal, although bridging occurred with an intersite distance of 10 to >or=63 nt. The close 10-nt spacing reduced the stability of dual-site binding, as competition for one site by a second CsrA dimer readily occurred. Both RNA-binding surfaces of a single CsrA protein were essential for efficient in vitro repression of a glgC'-'lacZ translational fusion that contains four CsrA target sites within the untranslated leader. Heterodimeric CsrA (HD-CsrA) containing a single R44A replacement, which was defective for binding at its mutant surface but bound RNA normally at its wild-type (WT) surface, was approximately 14-fold less effective at repression than homodimeric WT-CsrA. Furthermore, deletion of a CsrA target site of glgC that lies upstream from the Shine-Dalgarno sequence did not affect regulation by HD-CsrA but decreased regulation by WT-CsrA, confirming a regulatory role of dual-site binding. Finally, we propose a mechanism whereby a globular ribonucleoprotein complex is formed between CsrA and its noncoding RNA antagonist, CsrB. Because many target sites of CsrB are located closer together than is optimal for bridging, binding to nonadjacent sites should be energetically favored, causing multiple CsrA dimers to tether CsrB into the observed globular form rather than an extended CsrA-CsrB complex.

  4. Calicivirus 3C-Like Proteinase Inhibits Cellular Translation by Cleavage of Poly(A)-Binding Protein

    PubMed Central

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Green, Kim Y.; Lloyd, Richard E.

    2004-01-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CLpro) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CLpro PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3Cpro cleavage, while the FCV r3CLpro products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3Cpro cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CLpro was analyzed in HeLa cell translation extracts, and the presence of r3CLpro inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CLpro, like PV 3Cpro, mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation. PMID:15254188

  5. Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development.

    PubMed

    Gallie, Daniel R

    2017-01-01

    The poly(A)-binding protein (PABP) binds to the poly(A) tail of eukaryotic cellular mRNAs and contributes to their stability and translational efficiency. In plants, PABP is expressed from an unusually large gene family grouped into 3 classes that expanded during the evolution of land plants. Subsequent to expansion of the family, members diverged in their primary sequence and in expression. Further expansion of the family and divergence of its members in the Brassicaceae demonstrate the continued dynamic evolution of PABP in plants. In this study, the function of the widely-expressed class II PABP family members was examined to determine how individual class II members contribute to plant growth and development. Of the 3 class II PABP members, PAB2 and PAB4 contribute most to vegetative growth and vegetative-to-floral transition whereas PAB2, and the recently-evolved third class II member, PAB8, contribute to inflorescence and silique growth. Interestingly, although class I and class III PABP members are expressed specifically in reproductive organs, class II PABP members are also necessary for fertility in that the combinatorial loss of PAB2 and either PAB4 or PAB8 expression resulted in reduced fertility. Although all 3 class II members are required for protein expression, PAB4 contributes most to the steady-state level of a reporter mRNA and to protein expression. These findings suggest that class II PABP members are partially overlapping in function but also involved in distinct aspects of plant growth and development.

  6. Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate

    PubMed Central

    Byrne, Michael P.; Smith, Theresa J.; Montgomery, Vicki A.; Smith, Leonard A.

    1998-01-01

    Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 μg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 μg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of ≥0.5 μg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of ≥1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of ≤100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin. PMID:9746584

  7. ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.

    PubMed

    Costello, Joseph L; Castro, Inês G; Hacker, Christian; Schrader, Tina A; Metz, Jeremy; Zeuschner, Dagmar; Azadi, Afsoon S; Godinho, Luis F; Costina, Victor; Findeisen, Peter; Manner, Andreas; Islinger, Markus; Schrader, Michael

    2017-02-01

    Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering. © 2017 Costello et al.

  8. Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI.

    PubMed

    Baglia, F A; Jameson, B A; Walsh, P N

    1993-02-25

    Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with

  9. Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region.

    PubMed

    Wang, Guirong; Wu, Kongming; Liang, Gemei; Guo, Yuyuan

    2005-08-01

    Cadherins belong to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is related to insect's resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmembrane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respectively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recombinant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which located between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence

  10. Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex

    PubMed Central

    Gratia, Matthieu; Sarot, Emeline; Vende, Patrice; Charpilienne, Annie; Baron, Carolina Hilma; Duarte, Mariela

    2015-01-01

    ABSTRACT Through its interaction with the 5′ translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5′-capped and 3′-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3′ GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3′ end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation

  11. Arabidopsis Acyl-CoA-binding protein ACBP2 interacts with an ethylene-responsive element-binding protein, AtEBP, via its ankyrin repeats.

    PubMed

    Li, Hong-Ye; Chye, Mee-Len

    2004-01-01

    Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.

  12. Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G.

    PubMed Central

    Dominguez, D; Kislig, E; Altmann, M; Trachsel, H

    2001-01-01

    The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542-883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1-eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G(542-883) proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G(542-883) proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system. PMID:11256967

  13. NMR structure of rALF-Pm3, an anti-lipopolysaccharide factor from shrimp: model of the possible lipid A-binding site.

    PubMed

    Yang, Yinshan; Boze, Hélène; Chemardin, Patrick; Padilla, André; Moulin, Guy; Tassanakajon, Anchalee; Pugnière, Martine; Roquet, Françoise; Destoumieux-Garzón, Delphine; Gueguen, Yannick; Bachère, Evelyne; Aumelas, André

    2009-03-01

    The anti-lipopolysaccharide factor ALF-Pm3 is a 98-residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter as a folded and (15)N uniformly labeled rALF-Pm3 protein. Its 3D structure was established by NMR and consists of three alpha-helices packed against a four-stranded beta-sheet. The C(34)-C(55) disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF-Pm3 binds to LPS, lipid A and to OM-174, a soluble analogue of lipid A. Biophysical studies of rALF-Pm3/LPS and rALF-Pm3/OM-174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF-Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A-binding site of ALF-Pm3. Such a binding site, located on the ALF-Pm3 beta-sheet and involving seven charged residues, is well conserved in ALF-L from Limulus polyphemus and in ALF-T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that beta-hairpin synthetic peptides corresponding to ALF-L beta-sheet bind to LPS. Delineating lipid A-binding site of ALFs will help go further in the de novo design of new antibacterial or LPS-neutralizing drugs. (c) 2008 Wiley Periodicals, Inc.

  14. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    PubMed

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  15. Structure and Mechanism of Dimer-Monomer Transition of a Plant Poly(A)-Binding Protein upon RNA Interaction: Insights into Its Poly(A) Tail Assembly.

    PubMed

    Domingues, Mariane Noronha; Sforça, Mauricio Luis; Soprano, Adriana Santos; Lee, Jack; Souza, Tatiana de Arruda Campos Brasil de; Cassago, Alexandre; Portugal, Rodrigo Villares; Zeri, Ana Carolina de Mattos; Murakami, Mario Tyago; Sadanandom, Ari; Oliveira, Paulo Sergio Lopes de; Benedetti, Celso Eduardo

    2015-07-31

    Poly(A)-binding proteins (PABPs) play crucial roles in mRNA biogenesis, stability, transport and translational control in most eukaryotic cells. Although animal PABPs are well-studied proteins, the biological role, three-dimensional structure and RNA-binding mode of plant PABPs remain largely uncharacterized. Here, we report the structural features and RNA-binding mode of a Citrus sinensis PABP (CsPABPN1). CsPABPN1 has a domain architecture of nuclear PABPs (PABPNs) with a single RNA recognition motif (RRM) flanked by an acidic N-terminus and a GRPF-rich C-terminus. The RRM domain of CsPABPN1 displays virtually the same three-dimensional structure and poly(A)-binding mode of animal PABPNs. However, while the CsPABPN1 RRM domain specifically binds poly(A), the full-length protein also binds poly(U). CsPABPN1 localizes to the nucleus of plant cells and undergoes a dimer-monomer transition upon poly(A) interaction. We show that poly(A) binding by CsPABPN1 begins with the recognition of the RNA-binding sites RNP1 and RNP2, followed by interactions with residues of the β2 strands, which stabilize the dimer, thus leading to dimer dissociation. Like human PABPN1, CsPABPN1 also seems to form filaments in the presence of poly(A). Based on these data, we propose a structural model in which contiguous CsPABPN1 RRM monomers wrap around the RNA molecule creating a superhelical structure that could not only shield the poly(A) tail but also serve as a scaffold for the assembly of additional mRNA processing factors.

  16. Transcription Activation by NtcA in the Absence of Consensus NtcA-Binding Sites in an Anabaena Heterocyst Differentiation Gene Promoter

    PubMed Central

    Camargo, Sergio; Valladares, Ana; Flores, Enrique

    2012-01-01

    Heterocyst differentiation is orchestrated by the N control transcriptional regulator NtcA and the differentiation-specific factor HetR. In Anabaena sp. strain PCC 7120, the devBCA operon is expressed from two different promoters activated upon N stepdown. The distal devB promoter (transcription start point [TSP] located at position −704) represents a canonical class II NtcA-activated promoter, including a consensus NtcA-binding site centered 39.5 nucleotides upstream from the TSP. Transcription activation from a second TSP (−454) requires NtcA and is impaired in hetR mutants. In a wild-type background, three different DNA fragments, including both or each individual promoter, directed gfp expression localized mainly to proheterocysts and heterocysts. Expression was undetectable in an ntcA background and, for the fragment including the proximal promoter alone, also in a hetR background. In spite of the absence of consensus NtcA-binding sequences between the two TSPs, NtcA was shown to interact with this DNA region, and NtcA and its effector, 2-oxoglutarate, were necessary and sufficient for in vitro transcription from the −454 TSP. No HetR binding to the DNA or in vitro transcription from the proximal devB TSP promoted by HetR alone were detected. However, a moderate positive effect of HetR on NtcA binding to the DNA between the two devB TSPs was observed. The proximal devB promoter appears to represent a suboptimal NtcA-activated promoter for which HetR may act as a coactivator, with the physiological effect of restricting gene activation to conditions of prevalence of high NtcA and HetR levels, such as those taking place during heterocyst differentiation. PMID:22467790

  17. A hydrophobic loop in acyl-CoA binding protein is functionally important for binding to palmitoyl-coenzyme A: a molecular dynamics study.

    PubMed

    Vallejo, Diego F G; Grigera, J Raúl; Costabel, Marcelo D

    2008-04-01

    Acyl-CoA binding protein (ACBP) plays a key role in lipid metabolism, interacting via a partly unknown mechanism with high affinity with long chain fatty acyl-CoAs (LCFA-CoAs). At present there is no study of the microscopic way ligand binding is accomplished. We analyzed this process by molecular dynamics (MDs) simulations. We proposed a computational model of ligand, able to reproduce some evidence from nuclear magnetic resonance (NMR) data, quantitative time resolved fluorometry and X-ray crystallography. We found that a hydrophobic loop, not in the active site, is important for function. Besides, multiple sequence alignment shows hydrophobicity (and not the residues itselves) conservation.

  18. A novel RET rearrangement (ACBD5/RET) by pericentric inversion, inv(10)(p12.1;q11.2), in papillary thyroid cancer from an atomic bomb survivor exposed to high-dose radiation.

    PubMed

    Hamatani, Kiyohiro; Eguchi, Hidetaka; Koyama, Kazuaki; Mukai, Mayumi; Nakachi, Kei; Kusunoki, Yoichiro

    2014-11-01

    During analysis of RET/PTC rearrangements in papillary thyroid cancer (PTC) among atomic bomb survivors, a cDNA fragment of a novel type of RET rearrangement was identified in a PTC patient exposed to a high radiation dose using the improved 5' RACE method. This gene resulted from the fusion of the 3' portion of RET containing tyrosine kinase domain to the 5' portion of the acyl-coenzyme A binding domain containing 5 (ACBD5) gene, by pericentric inversion inv(10)(p12.1;q11.2); expression of the fusion gene was confirmed by RT-PCR. ACBD5 gene is ubiquitously expressed in various human normal tissues including thyroid. Full-length cDNA of the ACBD5-RET gene was constructed and then examined for tumorigenicity. Enhanced phosphorylation of ERK proteins in the MAPK pathway was observed in NIH3T3 cells transfected with expression vector encoding the full-length ACBD5/RET cDNA, while this was not observed in the cells transfected with empty expression vector. Stable NIH3T3 transfectants with ACBD5-RET cDNA induced tumor formation after their injection into nude mice. These findings suggest that the ACBD5-RET rearrangement is causatively involved in the development of PTC.

  19. The Golgi protein ACBD3 facilitates Enterovirus 71 replication by interacting with 3A

    PubMed Central

    Lei, Xiaobo; Xiao, Xia; Zhang, Zhenzhen; Ma, Yijie; Qi, Jianli; Wu, Chao; Xiao, Yan; Zhou, Zhuo; He, Bin; Wang, Jianwei

    2017-01-01

    Enterovirus 71 (EV71) is a human pathogen that causes hand, foot, mouth disease and neurological complications. Although EV71, as well as other enteroviruses, initiates a remodeling of intracellular membrane for genomic replication, the regulatory mechanism remains elusive. By screening human cDNA library, we uncover that the Golgi resident protein acyl-coenzyme A binding domain-containing 3 (ACBD3) serves as a target of the 3A protein of EV71. This interaction occurs in cells expressing 3A or infected with EV71. Genetic inhibition or deletion of ACBD3 drastically impairs viral RNA replication and plaque formation. Such defects are corrected upon restoration of ACBD3. In infected cells, EV71 3A redirects ACBD3, to the replication sites. I44A or H54Y substitution in 3A interrupts the binding to ACBD3. As such, viral replication is impeded. These results reveal a mechanism of EV71 replication that involves host ACBD3 for viral replication. PMID:28303920

  20. ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER

    PubMed Central

    Costello, Joseph L.; Hacker, Christian; Schrader, Tina A.; Zeuschner, Dagmar; Findeisen, Peter

    2017-01-01

    Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO–ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A–binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5–VAPB interaction regulates PO–ER associations. Moreover, we demonstrate that loss of PO–ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO–ER associations in mammalian cells and report a new function for ACBD5 in PO–ER tethering. PMID:28108524

  1. Ratcheted molecular-dynamics simulations identify efficiently the transition state of protein folding

    NASA Astrophysics Data System (ADS)

    Tiana, Guido; Camilloni, Carlo

    2012-12-01

    The atomistic characterization of the transition state (TS) is a fundamental step to improve the understanding of the folding mechanism and the function of proteins. From a computational point of view, the identification of the conformations that build out the transition state is particularly cumbersome, mainly because of the large computational cost of generating a statistically sound set of folding trajectories. Here we show that a biasing algorithm, based on the physics of the ratchet-and-pawl, can be used to approximate efficiently the transition state. The basic idea is that the algorithmic ratchet exerts a force on the protein when it is climbing the free-energy barrier, while it is inactive when it is descending. The transition state can be identified as the point of the trajectory where the ratchet changes regime. Besides discussing this strategy in general terms, we test it within a protein model whose transition state can be studied independently by plain molecular dynamics simulations. Finally, we show its power in explicit-solvent simulations, obtaining and characterizing a set of transition-state conformations for Acyl-Coenzyme A-Binding Protein (ACBP) and Chymotrypsin Inhibitor 2 (CI2).

  2. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    PubMed

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  3. Light-regulated Arabidopsis ACBP4 and ACBP5 encode cytosolic acyl-CoA-binding proteins that bind phosphatidylcholine and oleoyl-CoA ester.

    PubMed

    Xiao, Shi; Chen, Qin-Fang; Chye, Mee-Len

    2009-10-01

    In Arabidopsis thaliana, six genes encode acyl-CoA-binding proteins (ACBPs) that show conservation of an acyl-CoA-binding domain. These ACBPs display varying affinities for acyl-CoA esters, suggesting of different cellular roles. We have recently reported that three members (ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol by biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immuno-electron microscopy using ACBP-specific antibodies. In this study, we observed by Northern blot analysis that ACBP4 and ACBP5 mRNAs in rosettes were up-regulated by light and dampened-off in darkness, mimicking FAD7 which encodes omega-3-fatty acid desaturase, an enzyme involved in plastidial lipid metabolism. Results from in vitro binding assays indicate that recombinant ACBP4 and ACBP5 proteins bind [(14)C]oleoyl-CoA esters better than recombinant ACBP6, suggesting that light-regulated ACBP4 and ACBP5 encode cytosolic ACBPs that are potential candidates for the intracellular transport of oleoyl-CoA ester exported from the chloroplast to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids. Nonetheless, His-tagged ACBP4 and ACBP5 resemble ACBP6 in their ability to bind phosphatidylcholine suggesting that all three ACBPs are available for the intracellular transfer of phosphatidylcholine.

  4. Genome-wide mapping of TnrA-binding sites provides new insights into the TnrA regulon in Bacillus subtilis.

    PubMed

    Mirouze, Nicolas; Bidnenko, Elena; Noirot, Philippe; Auger, Sandrine

    2015-06-01

    Under nitrogen limitation conditions, Bacillus subtilis induces a sophisticated network of adaptation responses. More precisely, the B. subtilis TnrA regulator represses or activates directly or indirectly the expression of a hundred genes in response to nitrogen availability. The global TnrA regulon have already been identified among which some directly TnrA-regulated genes have been characterized. However, a genome-wide mapping of in vivo TnrA-binding sites was still needed to clearly define the set of genes directly regulated by TnrA. Using chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip), we now provide in vivo evidence that TnrA reproducibly binds to 42 regions on the chromosome. Further analysis with real-time in vivo transcriptional profiling, combined with results from previous reports, allowed us to define the TnrA primary regulon. We identified 35 promoter regions fulfilling three criteria necessary to be part of this primary regulon: (i) TnrA binding in ChIP-on-chip experiments and/or in previous in vitro studies; (ii) the presence of a TnrA box; (iii) TnrA-dependent expression regulation. In addition, the TnrA primary regulon delimitation allowed us to improve the TnrA box consensus. Finally, our results reveal new interconnections between the nitrogen regulatory network and other cellular processes.

  5. Cotranscriptional recruitment of the nuclear poly(A)-binding protein Pab2 to nascent transcripts and association with translating mRNPs.

    PubMed

    Lemieux, Caroline; Bachand, François

    2009-06-01

    Synthesis of the pre-mRNA poly(A) tail in the nucleus has important consequences on the translational activity of the mature mRNA in the cytoplasm. In most eukaryotes, nuclear polyadenylation of pre-mRNAs is thought to require the nuclear poly(A)-binding protein (PABP2/PABPN1) for poly(A) tail synthesis and ultimate length control. As yet, however, the extent of the association between PABP2 and the exported mRNA remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) assays to show that the fission yeast ortholog of mammalian PABP2 (Pab2) is cotranscriptionally recruited to active genes. Notably, the association of Pab2 to genes precedes that of a typical 3'-processing/polyadenylation factor, suggesting that Pab2 recruitment during the transcription cycle precedes polyadenylation. The inclusion of an RNase step in our ChIP and immunoprecipitation assays suggests that Pab2 is cotranscriptionally recruited via nascent mRNA ribonucleoprotein (mRNPs). Tandem affinity purification coupled with mass spectrometry also revealed that Pab2 associates with several ribosomal proteins as well as general translation factors. Importantly, whereas previous results suggest that the nuclear poly(A)-binding protein is not present on cytoplasmic mRNAs, we show that fission yeast Pab2 is associated with polysomes. Our findings suggest that Pab2 is recruited to nascent mRNPs during transcription and remains associated with translated mRNPs after nuclear export.

  6. The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications, including the methylation of glutamic acid.

    PubMed

    Low, Jason K K; Hart-Smith, Gene; Erce, Melissa A; Wilkins, Marc R

    2014-01-10

    Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR L-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.

  7. Involvement of the Acyl-CoA binding domain containing 7 in the control of food intake and energy expenditure in mice

    PubMed Central

    Lanfray, Damien; Caron, Alexandre; Roy, Marie-Claude; Laplante, Mathieu; Morin, Fabrice; Leprince, Jérôme; Tonon, Marie-Christine; Richard, Denis

    2016-01-01

    Acyl-CoA binding domain-containing 7 (Acbd7) is a paralog gene of the diazepam-binding inhibitor/Acyl-CoA binding protein in which single nucleotide polymorphism has recently been associated with obesity in humans. In this report, we provide converging evidence indicating that a splice variant isoform of the Acbd7 mRNA is expressed and translated by some POMC and GABAergic-neurons in the hypothalamic arcuate nucleus (ARC). We have demonstrated that the ARC ACBD7 isoform was produced and processed into a bioactive peptide referred to as nonadecaneuropeptide (NDN) in response to catabolic signals. We have characterized NDN as a potent anorexigenic signal acting through an uncharacterized endozepine G protein-coupled receptor and subsequently via the melanocortin system. Our results suggest that ACBD7-producing neurons participate in the hypothalamic leptin signalling pathway. Taken together, these data suggest that ACBD7-producing neurons are involved in the hypothalamic control exerted on food intake and energy expenditure by the leptin-melanocortin pathway. DOI: http://dx.doi.org/10.7554/eLife.11742.001 PMID:26880548

  8. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

    PubMed Central

    Mizuta, T R; Fukita, Y; Miyoshi, T; Shimizu, A; Honjo, T

    1993-01-01

    We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed. Images PMID:8493094

  9. Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

    SciTech Connect

    Wang Qishan Bag, Jnanankur

    2008-05-23

    Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO{sub 4}, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.

  10. Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

    PubMed

    Huang, Wenlin; Zhang, Zhongsheng; Ranade, Ranae M; Gillespie, J Robert; Barros-Álvarez, Ximena; Creason, Sharon A; Shibata, Sayaka; Verlinde, Christophe L M J; Hol, Wim G J; Buckner, Frederick S; Fan, Erkang

    2017-06-15

    Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC50 of 4nM. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Overproduction, purification and crystallization of a chondroitin sulfate A-binding DBL domain from a Plasmodium falciparum var2csa-encoded PfEMP1 protein

    SciTech Connect

    Higgins, Matthew K.

    2008-03-01

    A chondroitin sulfate A-binding DBL important in placental malaria has been overproduced, purified and crystallized. Diffraction data were collected to 1.9 Å resolution. The PfEMP1 proteins of the malaria parasite Plasmodium falciparum are inserted into the membrane of infected red blood cells, where they mediate adhesion to a variety of human receptors. The DBL domains of the var2csa-encoded PfEMP1 protein play a critical role in malaria of pregnancy, tethering infected cells to the surface of the placenta through interactions with the glycosaminoglycan carbohydrate chondroitin sulfate A (CSA). A CSA-binding DBL domain has been overproduced in a bacterial expression system, purified and crystallized. Native data sets extending to 1.9 Å resolution have been collected and phasing is under way.

  12. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

    PubMed

    Mizuta, T R; Fukita, Y; Miyoshi, T; Shimizu, A; Honjo, T

    1993-04-25

    We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed.

  13. Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

    PubMed

    Guerrero, Consuelo; Martín-Rufián, M; Reina, José J; Heredia, Antonio

    2006-01-01

    A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.

  14. A functional variant at miR-34a binding site in toll-like receptor 4 gene alters susceptibility to hepatocellular carcinoma in a Chinese Han population.

    PubMed

    Jiang, Zi-Cheng; Tang, Xian-Mei; Zhao, Ying-Ren; Zheng, Lei

    2014-12-01

    Toll-like receptor 4 (TLR4) plays a key role in prompting the innate or immediate response. A growing body of evidence suggests that genetic variants of TLR4 gene were associated with the development of cancers. This study aimed to investigate the relationship of a functional variant (rs1057317) at microRNA-34a (miR-34a) binding site in toll-like receptor 4 gene and the risk of hepatocellular carcinoma. A single center-based case-control study was conducted. In this study, the polymerase chain reaction (PCR) and direct sequencing were used to genotype sequence variants of TLR4 in 426 hepatocellular carcinoma cases and 438 controls. The modification of rs1057317 on the binding of hsa-miR-34a to TLR4 messenger RNA (mRNA) was measured by luciferase activity assay. Individuals carrying the AA genotypes for the rs1057317 were associated significantly with increased risk of hepatocellular carcinoma comparing with those carrying wild-type homozygous CC genotypes (adjusted odds ratio [OR] by sex and age, from 1.116 to 2.452, P = 0.013). The activity of the reporter vector was lower in the reporter vector carrying C allele than the reporter vector carrying A allele. Furthermore, the expression of TLR4 was detected in the peripheral blood mononucleated cell of hepatocellular carcinoma (HCC) patients, suggesting that mRNA and protein levels of TLR4 might be associated with SNP rs1057317. Collectively, these results suggested that the risk of hepatocellular carcinoma was associated with a functional variant at miR-34a binding site in toll-like receptor 4 gene. miR-34a/TLR4 axis may play an important role in the development of hepatocellular carcinoma.

  15. The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties.

    PubMed

    Brutinel, Evan D; King, Jessica M; Marsden, Anne E; Yahr, Timothy L

    2012-05-01

    Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. Each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two helix-turn-helix (HTH) DNA-binding motifs of an ExsA monomer. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. In the present study, we demonstrate that site 2 in the P(exsC) promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer. Likewise, the second HTH interacts with an adenine residue in binding site 2. Although several candidate GnC sequences are also present in site 2 of the P(exsD), P(exoT), and P(pcrG) promoters, the GnC sequences were not required for ExsA-dependent transcription or ExsA binding. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Based on these data, we propose that the manner in which ExsA interacts with binding site 2 at the P(exsC) promoter is distinct from the interactions occurring at other promoters.

  16. Acyl-CoA binding protein expression is fiber type- specific and elevated in muscles from the obese insulin-resistant Zucker rat.

    PubMed

    Franch, Jesper; Knudsen, Jens; Ellis, Bronwyn A; Pedersen, Preben K; Cooney, Gregory J; Jensen, Jørgen

    2002-02-01

    Accumulation of acyl-CoA is hypothesized to be involved in development of insulin resistance. Acyl-CoA binds to acyl-CoA binding protein (ACBP) with high affinity, and therefore knowledge about ACBP concentration is important for interpreting acyl-CoA data. In the present study, we used a sandwich enzyme-linked immunosorbent assay to quantify ACBP concentration in different muscle fiber types. Furthermore, ACBP concentration was compared in muscles from lean and obese Zucker rats. Expression of ACBP was highest in the slow-twitch oxidative soleus muscle and lowest in the fast-twitch glycolytic white gastrocnemius (0.46 +/- 0.02 and 0.16 +/- 0.005 microg/mg protein, respectively). Expression of ACBP was soleus > red gastrocnemius > extensor digitorum longus > white gastrocnemius. Similar fiber type differences were found for carnitine palmitoyl transferase (CPT)-1, and a correlation was observed between ACBP and CPT-1. Muscles from obese Zucker rats had twice the triglyceride content, had approximately twice the long-chain acyl CoA content, and were severely insulin resistant. ACBP concentration was approximately 30% higher in all muscles from obese rats. Activities of CPT-1 and 3-hydroxy-acyl-CoA dehydrogenase were increased in muscles from obese rats, whereas citrate synthase activity was similar. In conclusion, ACBP expression is fiber type-specific with the highest concentration in oxidative muscles and the lowest in glycolytic muscles. The 90% increase in the concentration of acyl-CoA in obese Zucker muscle compared with only a 30% increase in the concentration of ACBP supports the hypothesis that an increased concentration of free acyl-CoA is involved in the development of insulin resistance.

  17. Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

    PubMed

    Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2014-08-17

    Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

  18. The bent conformation of poly(A)-binding protein induced by RNA-binding is required for its translational activation function

    PubMed Central

    Hong, Ka Young; Lee, Seung Hwan; Gu, Sohyun; Kim, Eunah; An, Sihyeon; Kwon, Junyoung; Jang, Sung Key

    2017-01-01

    ABSTRACT A recent study revealed that poly(A)-binding protein (PABP) bound to poly(A) RNA exhibits a sharply bent configuration at the linker region between RNA-recognition motif 2 (RRM2) and RRM3, whereas free PABP exhibits a highly flexible linear configuration. However, the physiological role of the bent structure of mRNA-bound PABP remains unknown. We investigated a role of the bent structure of PABP by constructing a PABP variant that fails to form the poly(A)-dependent bent structure but maintains its poly(A)-binding activity. We found that the bent structure of PABP/poly(A) complex is required for PABP's efficient interaction with eIF4G and eIF4G/eIF4E complex. Moreover, the mutant PABP had compromised translation activation function and failed to augment the formation of 80S translation initiation complex in an in vitro translation system. These results suggest that the bent conformation of PABP, which is induced by the interaction with 3′ poly(A) tail, mediates poly(A)-dependent translation by facilitating the interaction with eIF4G and the eIF4G/eIF4E complex. The preferential binding of the eIF4G/eIF4E complex to the bent PABP/poly(A) complex seems to be a mechanism discriminating the mRNA-bound PABPs participating in translation from the idling mRNA-unbound PABPs. PMID:28095120

  19. Purification and characterization of the human SR 31747A-binding protein. A nuclear membrane protein related to yeast sterol isomerase.

    PubMed

    Jbilo, O; Vidal, H; Paul, R; De Nys, N; Bensaid, M; Silve, S; Carayon, P; Davi, D; Galiègue, S; Bourrié, B; Guillemot, J C; Ferrara, P; Loison, G; Maffrand, J P; Le Fur, G; Casellas, P

    1997-10-24

    SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a Mr 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10, 047 being a more potent competitor than (-)-SKF 10,047. Scatchard plot analysis revealed Kd values of 7.1 nM and 0.15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.

  20. Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay.

    PubMed

    Silva, Ana Luísa; Ribeiro, Patrícia; Inácio, Angela; Liebhaber, Stephen A; Romão, Luísa

    2008-03-01

    mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.

  1. Bridging the divide between sensory integration and binding theory: Using a binding-like neural synchronization mechanism to model sensory enhancements during multisensory interactions.

    PubMed

    Billock, Vincent A; Tsou, Brian H

    2014-07-01

    Neural information combination problems are ubiquitous in cognitive neuroscience. Two important disciplines, although conceptually similar, take radically different approaches to these problems. Sensory binding theory is largely grounded in synchronization of neurons responding to different aspects of a stimulus, resulting in a coherent percept. Sensory integration focuses more on the influences of the senses on each other and is largely grounded in the study of neurons that respond to more than one sense. It would be desirable to bridge these disciplines, so that insights gleaned from either could be harnessed by the other. To link these two fields, we used a binding-like oscillatory synchronization mechanism to simulate neurons in rattlesnake that are driven by one sense but modulated by another. Mutual excitatory coupling produces synchronized trains of action potentials with enhanced firing rates. The same neural synchronization mechanism models the behavior of a population of cells in cat visual cortex that are modulated by auditory activation. The coupling strength of the synchronizing neurons is crucial to the outcome; a criterion of strong coupling (kept weak enough to avoid seriously distorting action potential amplitude) results in intensity-dependent sensory enhancement-the principle of inverse effectiveness-a key property of sensory integration.

  2. A spider toxin, ω-agatoxin IV A, binds to fixed as well as living tissues: cytochemical visualization of P/Q-type calcium channels.

    PubMed

    Nakanishi, Setsuko

    2016-08-01

    ω-Agatoxin IV A, a peptidyl toxin from Agelenopsis aperta venom, selectively binds to voltage-gated P/Q-type calcium channels. ω-Agatoxin IV A has been used as a selective tool in pharmacological and electrophysiological studies. Visualization of P/Q-type calcium channels has previously been accomplished using biotin-conjugated ω-Agatoxin IV A in freshly prepared mouse cerebellar and hippocampal slices (Nakanishi et al, J. Neurosci. Res., 41: , 532, 1995). Here biotinylated ω-agatoxin IV A was applied to transcardially fixed brain slices prepared with various fixatives. ω-Agatoxin IV A did not bind to fixed tissues from P/Q-type calcium channel knockout mice, confirming that binding to normal, fixed tissues was not an artifact. Using transmission electron microscopy, locations of biotinylated ω-agatoxin IV A binding sites visualized with gold-conjugated streptavidin showed a similar pattern to those visualized with antibody. The ability of biotinylated ω-agatoxin IV A to bind to fixed tissue provides a new cytochemical technique to study molecular architecture of synapses.

  3. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

    SciTech Connect

    Wang Qishan; Bag, Jnanankur . E-mail: jbag@uoguelph.ca

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  4. A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine.

    PubMed

    Yurchenko, Olga P; Nykiforuk, Cory L; Moloney, Maurice M; Ståhl, Ulf; Banaś, Antoni; Stymne, Sten; Weselake, Randall J

    2009-09-01

    The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

  5. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

    PubMed

    Ray, Swagat; Anderson, Emma C

    2016-03-03

    The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

  6. PAB-1, a Caenorhabditis elegans poly(A)-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    PubMed

    Ko, Sunhee; Kawasaki, Ichiro; Shim, Yhong-Hee

    2013-01-01

    Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  7. An evolutionarily conserved interaction of tumor suppressor protein Pdcd4 with the poly(A)-binding protein contributes to translation suppression by Pdcd4.

    PubMed

    Fehler, Olesja; Singh, Priyanka; Haas, Astrid; Ulrich, Diana; Müller, Jan P; Ohnheiser, Johanna; Klempnauer, Karl-Heinz

    2014-01-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) has been implicated in the translational regulation of specific mRNAs, however, the identities of the natural Pdcd4 target mRNAs and the mechanisms by which Pdcd4 affects their translation are not well understood. Pdcd4 binds to the eukaryotic translation initiation factor eIF4A and inhibits its helicase activity, which has suggested that Pdcd4 suppresses translation initiation of mRNAs containing structured 5'-untranslated regions. Recent work has revealed a second inhibitory mechanism, which is eIF4A-independent and involves direct RNA-binding of Pdcd4 to the target mRNAs. We have now identified the poly(A)-binding protein (PABP) as a novel direct interaction partner of Pdcd4. The ability to interact with PABP is shared between human and Drosophila Pdcd4, indicating that it has been highly conserved during evolution. Mutants of Pdcd4 that have lost the ability to interact with PABP fail to stably associate with ribosomal complexes in sucrose density gradients and to suppress translation, as exemplified by c-myb mRNA. Overall, our work identifies PABP as a novel functionally relevant Pdcd4 interaction partner that contributes to the regulation of translation by Pdcd4.

  8. Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: A vegetal PABC domain

    PubMed Central

    Siddiqui, Nadeem; Kozlov, Guennadi; D’Orso, Iván; Trempe, Jean-François; Gehring, Kalle

    2003-01-01

    PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five α-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes. PMID:12930992

  9. A genetic variant of miR-148a binding site in the SCRN1 3′-UTR is associated with susceptibility and prognosis of gastric cancer

    PubMed Central

    Song, Peng; Zhu, Haixia; Zhang, Dong; Chu, Haiyan; Wu, Dongmei; Kang, Meiyun; Wang, Meilin; Gong, Weida; Zhou, Jianwei; Zhang, Zhengdong; Zhao, Qinghong

    2014-01-01

    Single nucleotide polymorphisms (SNPs) in the 3′-untranslated regions targeted by putative mircoRNA can change its binding strength, affecting the susceptibility and prognosis of cancer. We aimed to investigate the associations between SNPs within miR-148a binding sites and gastric cancer (GC) risk and prognosis. Using bioinformatics tools, we selected two SNPs (SCRN1 rs6976789 and PDYN rs2235749) located in miR-148a target sites. We genotyped the two SNPs in a case-control study comprising 753 GC patients and 949 cancer-free subjects. We found a significantly increased risk of GC associated with the SCRN1 rs6976789 C>T polymorphism [adjusted OR = 1.25, 95% confidence interval (CI) = 1.02–1.53; CT/TT vs. CC]. However, no significant association was found between the PDYN rs2235749 and GC risk in all genetic models. Furthermore, we evaluated whether SCRN1 rs6976789 affected the survival of GC patients. Results showed that individuals with SCRN1 rs6976789 TT genotype had poorer overall survival compared with those carried CC/CT genotypes in intestinal-type GC (adjusted HR = 2.47, 95% CI = 1.21–5.05). Luciferase report assay showed that the rs6976789 variant T allele influenced the binding ability of miR-148a. Our results suggested that the SCRN1 rs6976789 polymorphism may play an important role in the GC development and progression. PMID:25399950

  10. Escherichia coli phage-shock protein A (PspA) binds to membrane phospholipids and repairs proton leakage of the damaged membranes.

    PubMed

    Kobayashi, Ryuji; Suzuki, Toshiharu; Yoshida, Masasuke

    2007-10-01

    Escherichia coli phage-shock protein A (PspA), a 25.3 kDa peripheral membrane protein, is induced under the membrane stress conditions and is assumed to help maintain membrane potential. Here, we report that purified PspA, existing as a large oligomer, is really able to suppress proton leakage of the membranes. This was demonstrated for membrane vesicles prepared from the PspA-lacking E. coli mutants, and for membrane vesicles damaged by ethanol and Triton X-100 prepared from the mutant and the wild-type cells. PspA also suppressed proton leakage of damaged liposomes made from E. coli total lipids. Furthermore, we found that PspA bound preferentially to liposomes containing phosphatidylserine and phosphatidylglycerol. All these effects were not observed for monomer PspA that was prepared by refolding of urea-denatured PspA. These results indicate that oligomers of PspA bind to membrane phospholipids and suppress proton leakage.

  11. Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

    PubMed Central

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F.

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis. PMID:23166793

  12. Measurement of dissolved reactive phosphorus in water with polyquaternary ammonium salt as a binding agent in diffusive gradients in thin-films technique.

    PubMed

    Chen, Hong; Zhang, Meng-Han; Gu, Jia-Li; Zhao, Gang; Zhang, Yu; Li, Jian-Rong

    2014-12-17

    Diffusive gradients in thin-films (DGT) sampler with a polyquaternary ammonium salt (PQAS) aqueous solution as a binding phase and a dialysis membrane as a diffusive phase (PQAS DGT) was developed for the measurement of dissolved reactive phosphorus (DRP) in water. The performance of PQAS DGT was not dependent upon pH 3-10 and ionic strength from 1 × 10(-4) to 1 mol L(-1). The effective binding capacity of PQAS DGT containing 2.0 mL of 0.050 mol L(-1) PQAS solution was estimated as 9.9 μg cm(-2). The measurement of DRP in a synthetic solution by PQAS DGT over a 48 h deployment period demonstrated high consistency with the concentration of DRP in the synthetic solution measured directly by the ammonium molybdate spectrophotometric method. Field deployments of PQAS DGT samplers allowed for accurate measurement of the DRP concentration in situ. The advantages of PQAS DGT include no requirement of the elution steps and direct concentration measurements of the binding phase.

  13. The recruitment of the Saccharomyces cerevisiae poly(A)-binding protein into stress granules: new insights into the contribution of the different protein domains.

    PubMed

    Brambilla, Marco; Martani, Francesca; Branduardi, Paola

    2017-09-01

    The Saccharomyces cerevisiae poly(A)-binding protein Pab1 is a modular protein composed of four RNA recognition motifs (RRM), a proline-rich domain (P) and a C-terminus. Thanks to this modularity, Pab1 is involved in different interactions that regulate many aspects of mRNA metabolism, including the assembly of stress granules. In this work, we analyzed the contribution of each domain for the recruitment of the protein within stress granules by comparing the intracellular distribution of synthetic Pab1-GFP variants, lacking one or more domains, with the localization of the endogenous mCherry-tagged Pab1. Glucose starvation and heat shock were used to trigger the formation of stress granules. We found that Pab1 association into these aggregates relies mainly on RRMs, whose number is important for an efficient recruitment of the protein. Interestingly, although the P and C domains do not directly participate in Pab1 association to stress granules, their presence strengthens or decreases, respectively, the distribution of synthetic Pab1 lacking at least one RRM into these aggregates. In addition to describing the contribution of domains in determining Pab1 association within stress granules, the outcomes of this study suggest the modularity of Pab1 as an attractive platform for synthetic biology approaches aimed at rewiring mRNA metabolism. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Trinucleotide expansions leading to an extended poly-L-alanine segment in the poly (A) binding protein PABPN1 cause fibril formation.

    PubMed

    Scheuermann, Till; Schulz, Barbe; Blume, Alfred; Wahle, Elmar; Rudolph, Rainer; Schwarz, Elisabeth

    2003-12-01

    The nuclear poly(A) binding protein (PABPN1) stimulates poly(A) polymerase and controls the lengths of poly(A) tails during pre-mRNA processing. The wild-type protein possesses 10 consecutive Ala residues immediately after the start methionine. Trinucleotide expansions in the coding sequence result in an extension of the Ala stretch to maximal 17 Ala residues in total. Individuals carrying the trinucleotide expansions suffer from oculopharyngeal muscular dystrophy (OPMD). Intranuclear inclusions consisting predominantly of PABPN1 have been recognized as a pathological hallmark of the genetic disorder. To elucidate the molecular events that lead to disease, recombinant PABPN1, and N-terminal fragments of the protein with varying poly-L-alanine stretches were analyzed. As the full-length protein displayed a strong tendency to aggregate into amorphous deposits, soluble N-terminal fragments were also studied. Expansion of the poly-L-alanine sequence to the maximal length observed in OPMD patients led to an increase of alpha-helical structure. Upon prolonged incubation the protein was found in fibrils that showed all characteristics of amyloid-like fibers. The lag-phase of fibril formation could be reduced by seeding. Structural analysis of the fibrils indicated antiparallel beta-sheets.

  15. Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13762 rat mammary ascites tumor cells

    SciTech Connect

    Vanderpuye, L.A.; Carraway, C.A.C.; Carraway, K.L. )

    1988-10-01

    Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. Phalloidin shift analyses on velocity sedimentation gradients of Triton X-100 extracts of ({sup 3}H)-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Since the Triton-insoluble membrane residue is enriched in actin-containing transmembrane complex, which contains a different glycoprotein, the authors suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

  16. Arabidopsis acyl-CoA-binding proteins ACBP4 and ACBP5 are subcellularly localized to the cytosol and ACBP4 depletion affects membrane lipid composition.

    PubMed

    Xiao, Shi; Li, Hong-Ye; Zhang, Jiao-Ping; Chan, Suk-Wah; Chye, Mee-Len

    2008-12-01

    In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they display varying affinities for acyl-CoA esters. Recombinant ACBP4 and ACBP5 have been shown to bind oleoyl-CoA esters in vitro. In this study, the subcellular localizations of ACBP4 and ACBP5 were determined by biochemical fractionation followed by western blot analyses using anti-ACBP4 and anti-ACBP5 antibodies and immuno-electron microscopy. Confocal microscopy of autofluorescence-tagged ACBP4 and ACBP5, expressed transiently in onion epidermal cells and in transgenic Arabidopsis, confirmed their expression in the cytosol. Taken together, ACBP4 and ACBP5 are available in the cytosol to bind and transfer cytosolic oleoyl-CoA esters. Lipid profile analysis further revealed that an acbp4 knockout mutant showed decreases in membrane lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) while acbp4-complemented lines attained levels similar to wild type, suggesting that ACBP4 plays a role in the biosynthesis of membrane lipids including galactolipids and phospholipids.

  17. HBXIP, a binding protein of HBx, regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity.

    PubMed

    Fei, Hongrong; Zhou, Yunsheng; Li, Ruotong; Yang, Mingfeng; Ma, Jian; Wang, Fengze

    2017-03-04

    To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.

  18. Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs

    PubMed Central

    Muniz, Lisa; Davidson, Lee

    2015-01-01

    Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3′-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing. PMID:25896913

  19. Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay

    PubMed Central

    Fatscher, Tobias; Gehring, Niels H.

    2016-01-01

    Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3′ UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3′ UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3′ UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing β-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD. PMID:27874031

  20. Loss of the acyl-CoA binding protein (Acbp) results in fatty acid metabolism abnormalities in mouse hair and skin.

    PubMed

    Lee, Lance; DeBono, C Anthony; Campagna, Dean R; Young, David C; Moody, D Branch; Fleming, Mark D

    2007-01-01

    Proper fatty acid metabolism is critical for hair and skin development and maintenance. The acyl-CoA binding protein (Acbp) is a widely expressed protein that binds long-chain fatty acyl-CoA esters and plays a role in fatty acyl-CoA transport and pool formation. However, loss of function of Acbp in the whole animal has not been investigated. Here, we show that deletion of Acbp in mouse results in sebocyte hyperplasia and sparse, matted hair with a greasy appearance. Consistent with these gross abnormalities, Acbp is highly expressed in the pilosebaceous units of mouse skin as determined by Northern analysis and in situ hybridization. Loss of Acbp also results in fatty acid metabolism abnormalities, with hair lipid profiles showing altered levels of triacylglycerols and nearly co-migrating lipids. These data suggest that Acbp plays a role in triacylglycerol biosynthesis, and that regulation of this process is important for proper hair and skin development and maintenance in the mouse.

  1. Maturation and activity of sterol regulatory element binding protein 1 is inhibited by acyl-CoA binding domain containing 3.

    PubMed

    Chen, Yong; Patel, Vishala; Bang, Sookhee; Cohen, Natalie; Millar, John; Kim, Sangwon F

    2012-01-01

    Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

  2. Wheat germ poly(A) binding protein enhances the binding affinity of eukaryotic initiation factor 4F and (iso)4F for cap analogues.

    PubMed

    Wei, C C; Balasta, M L; Ren, J; Goss, D J

    1998-02-17

    Most eukaryotic mRNAs contain a 5' cap (m7GppX) and a 3' poly(A) tail to increase synergistically the translational efficiency. Recently, the poly(A) binding protein (PABP) and cap-binding protein, eIF-4F, were found to interact [Le et al. (1997) J. Biol. Chem. 272, 16247-16255; Tarun and Sachs (1996) EMBO J. 15, 7168-7177]. These data suggest that PABP may exert its effect on translational efficiency either by increasing the formation of initiation factor-mRNA complex or by enhancing ribosome recycling. To investigate the functional consequences of these interactions, the fluorescent cap analogue, ant-m7GTP, which is an environmentally sensitive fluorescent probe [Ren and Goss (1996) Nucleic Acids Res. 24, 3629-3634] was used to investigate the cap-binding affinity. Our data show that the binding of eIF-(iso)4F or eIF-4F to cap analogue enhanced their binding affinity toward PABP approximately 40-fold. Similarly, the eIF-4F/PABP or eIF-(iso)4F/PABP complexes show a 40-fold enhancement of cap analogue binding as compared to eIF-4F or eIF-(iso)4F alone. At least part of the enhancement of the translational initiation by PABP can be accounted for by direct changes in cap-binding affinity. The interactions of these components also suggest a mechanism whereby the poly(A) tail is brought into close proximity with m7G cap. This effect was examined by fluorescence energy transfer, and it was determined that the PABP/eIF-4F complex could bind both poly(A) and 5' cap simultaneously.

  3. EphrinA4 mimetic peptide targeted to EphA binding site impairs the formation of long-term fear memory in lateral amygdala

    PubMed Central

    Dines, M; Lamprecht, R

    2014-01-01

    Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders. PMID:25268254

  4. EphrinA4 mimetic peptide targeted to EphA binding site impairs the formation of long-term fear memory in lateral amygdala.

    PubMed

    Dines, M; Lamprecht, R

    2014-09-30

    Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathologies conditions such as phobias and posttraumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). EphrinA4 and its cognate Eph receptors are intimately involved in regulating neuronal morphogenesis, synaptic transmission and plasticity. To assess possible roles of ephrinA4 in fear memory formation we designed and used a specific inhibitory ephrinA4 mimetic peptide (pep-ephrinA4) targeted to EphA binding site. We show that this peptide, composed of the ephrinA4 binding domain, interacts with EphA4 and inhibits ephrinA4-induced phosphorylation of EphA4. Microinjection of the pep-ephrinA4 into rat LA 30 min before training impaired long- but not short-term fear conditioning memory. Microinjection of a control peptide derived from a nonbinding E helix site of ephrinA4, that does not interact with EphA, had no effect on fear memory formation. Microinjection of pep-ephrinA4 into areas adjacent to the amygdala had no effect on fear memory. Acute systemic administration of pep-ephrinA4 1 h after training also impaired long-term fear conditioning memory formation. These results demonstrate that ephrinA4 binding sites in LA are essential for long-term fear memory formation. Moreover, our research shows that ephrinA4 binding sites may serve as a target for pharmacological treatment of fear and anxiety disorders.

  5. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

    PubMed

    Soto, Manuel; Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M; Martín, M Elena; Alonso, Carlos; Coelho, Eduardo A F; Barral, Aldina; Barral-Netto, Manoel; Iborra, Salvador

    2015-05-01

    Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

  6. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Morita, Takahiro; Satoh, Ryosuke; Umeda, Nanae; Kita, Ayako; Sugiura, Reiko

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Stress granules (SGs) as a mechanism of doxorubicin tolerance. Black-Right-Pointing-Pointer We characterize the role of stress granules in doxorubicin tolerance. Black-Right-Pointing-Pointer Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. Black-Right-Pointing-Pointer Doxorubicin promotes SG formation when combined with heat shock. Black-Right-Pointing-Pointer Doxorubicin regulates stress granule assembly independent of eIF2{alpha} phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1{sup +}, which encodes a multi-KH type RNA-binding protein, and pab1{sup +}, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1{sup +} and pab1{sup +} genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2{alpha}, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2{alpha} phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  7. Molecular cloning and chromosomal localization of a pseudogene related to the human Acyl-CoA binding protein/diazepam binding inhibitor

    SciTech Connect

    Gersuk, V.H.; Rose, T.M.; Todaro, G.J.

    1995-01-20

    The acyl-CoA binding protein (ACBP) and the diazepam binding inhibitor (DBI) or endozepine are independent isolates of a single 86-amino-acid, 10-kDa protein. ACBP/DBI is highly conserved between species and has been identified in several diverse organisms, including human, cow, rat, frog, duck, insects, plants, and yeast. Although the genomic locus has not yet been cloned in humans, complementary DNA clones with different 5{prime} ends have been isolated and characterized. These cDNA clones appear to be encoded by a single gene. However, Southern blot analyses, in situ hybridizations, and somatic cell hybrid chromosomal mapping all suggest that there are multiple ACBP/DBI-related sequences in the genome. To identify potential members of this gene family, degenerate oligonucleotides corresponding to highly conserved regions of ACBP/DBI were used to screen a human genomic DNA library using the polymerase chain reaction. A novel gene, DBIP1, that is closely related to ACBP/DBI but is clearly distinct was identified. DBIP1 bears extensive sequence homology to ACBP/DBI but lacks the introns predicted by rat and duck genomic sequence studies. A 1-base deletion in the coding region results in a frameshift and, along with the absence of introns and the lack of a detectable transcript, suggests that DBIP1 is a pseudogene. ACBP/DBI has previously been mapped to chromosome 2, although this was recently disputed, and a chromosome 6 location was suggested. We show that ACBP/DBI is correctly placed on chromosome 2 and that the gene identified on chromosome 6 is DBIP1. 33 refs., 3 figs., 1 tab.

  8. Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling*

    PubMed Central

    Bodman, James A. R.; Yang, Yang; Logan, Michael R.; Eitzen, Gary

    2015-01-01

    Rho GTPases are molecular switches that modulate a variety of cellular processes, most notably those involving actin dynamics. We have previously shown that yeast vacuolar membrane fusion requires re-organization of actin filaments mediated by two Rho GTPases, Rho1p and Cdc42p. Cdc42p initiates actin polymerization to facilitate membrane tethering; Rho1p has a role in the late stages of vacuolar fusion, but its mode of action is unknown. Here, we identified eEF1A as a vacuolar Rho1p-interacting protein. eEF1A (encoded by the TEF1 and TEF2 genes in yeast) is an aminoacyl-tRNA transferase needed during protein translation. eEF1A also has a second function that is independent of translation; it binds and organizes actin filaments into ordered cable structures. Here, we report that eEF1A interacts with Rho1p via a C-terminal subdomain. This interaction occurs predominantly when both proteins are in the GDP-bound state. Therefore, eEF1A is an atypical downstream effector of Rho1p. eEF1A does not promote vacuolar fusion; however, overexpression of the Rho1p-interacting subdomain affects vacuolar morphology. Vacuoles were destabilized and prone to leakage when treated with the eEF1A inhibitor narciclasine. We propose a model whereby eEF1A binds to Rho1p-GDP on the vacuolar membrane; it is released upon Rho1p activation and then bundles actin filaments to stabilize fused vacuoles. Therefore, the Rho1p-eEF1A complex acts to spatially localize a pool of eEF1A to vacuoles where it can readily organize F-actin. PMID:25561732

  9. Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells

    PubMed Central

    Duren, Ryan P.; Boudreaux, Seth P.; Conneely, Orla M.

    2016-01-01

    Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells. PMID:26938745

  10. Evolutionary history exposes radical diversification among classes of interaction partners of the MLLE domain of plant poly(A)-binding proteins.

    PubMed

    Jiménez-López, Domingo; Bravo, Jaime; Guzmán, Plinio

    2015-09-16

    Poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that have important functions in the regulation of translation and the control of mRNA stability in eukaryotes. Most PABPs encode a C-terminal domain known as the MLLE domain (previously PABC or CTC), which can mediate protein interactions. In earlier work we identified and predicted that four classes of MLLE-interacting proteins were present in Arabidopsis thaliana, which we named CID A, B, C, and D. These proteins encode transcription-activating domains (CID A), the Lsm and LsmAD domains of ataxin-2 (CID B), the CUE and small MutS-related domains (CID C), and two RNA recognition domains (CID D). We recently found that a novel class that lacks the LsmAD domain is present in CID B proteins. We extended our analysis to other classes of CIDs present in the viridiplantae. We found that novel variants also evolved in classes CID A and CID C. A specific transcription factor domain is present in a distinct lineage in class A, and a variant that lacks at least two distinct domains was also identified in a divergent lineage in class C. We did not detect any variants in Class D CIDs. This class often consists of four to six highly conserved RNA-binding proteins, which suggests that major redundancy is present in this class. CIDs are likely to operate as components of posttranscriptional regulatory assemblies. The evident diversification of CIDs may be neutral or may be important for plant adaptation to the environment and for acquisition of specific traits during evolution. The fact that CIDs subclasses are maintained in early lineages suggest that a presumed interference between duplicates was resolved, and a defined function for each subclass was achieved.

  11. PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, II-1, and Po proline-rich proteins

    SciTech Connect

    Azen, E.A.; Amberger, E.; Niece, R.L.

    1996-01-01

    Six closely linked PRP (proline-rich protein) genes code for many salivary PRPs that show frequent length and null variants. From determined protein sequences and DNA sequence analysis of variant alleles, we here report the coding and molecular basis for Con (concanavalin A-binding) and Po (parotid {open_quotes}o{close_quotes}) protein polymorphisms. The Con1 glycoprotein is encoded in exon 3 of a PRB2 allele (PRB2L CON1+) with a potential N-linked glycosylation site. Because of a probable gene conversion encompassing {ge}684 bp of DNA, the {open_quotes}PRB2-like{close_quotes} Con2 glycoprotein is encoded in exon 3 of a PRB1 allele (PRB1M CON2+) with a potential glycosylation site. The PmF protein is also encoded in the PRB1M CON2+ allele, thus explaining the previously reported association between Con2 and PmF proteins. A PRB2L CON1-allele contains a single nt missense change [TCT(Ser){yields}CCT(Pro)] that abolishes the potential N-linked glycosylation site (NKS{yields}NKP) in the Con1 protein, and this explains the Con-type. The Po protein and a glycoprotein (II-1) are encoded in the PRB4 gene, and both proteins are absent in the presence of a mutation in the PRB4M PO- allele that contains a single nt change (G{yields}C) at the +1 invariant position of the intron 3 5{prime} donor splice site. The genetically determined absence of the II-1 glycoprotein leads to altered in vitro binding of Streptococcus sanguis 10556 to salivary proteins, which suggests a biological consequence for null mutations of the PRB4 gene. 33 refs., 7 figs., 1 tab.

  12. Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres.

    PubMed

    Alonso, Alicia; Mahmood, Radma; Li, Shulan; Cheung, Fanny; Yoda, Kinya; Warburton, Peter E

    2003-10-15

    Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres. A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation. Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA. Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal approximately 7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of approximately 7 Mb. DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3-13q33.1. The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of approximately 130, 215 and 275 kb within an approximately 6.5 Mb region. Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere 'hotspot' in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres. The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

  13. Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior.

    PubMed

    Fronz, Katharina; Otto, Silke; Kölbel, Knut; Kühn, Uwe; Friedrich, Henning; Schierhorn, Angelika; Beck-Sickinger, Annette G; Ostareck-Lederer, Antje; Wahle, Elmar

    2008-07-18

    The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

  14. The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay.

    PubMed

    Fatscher, Tobias; Boehm, Volker; Weiche, Benjamin; Gehring, Niels H

    2014-10-01

    Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3' UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR. © 2014 Fatscher et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. The Crystal Structure of PPIL1 Bound to Cyclosporine A Suggests a Binding Mode for a Linear Epitope of the SKIP Protein

    PubMed Central

    Stegmann, Christian M.; Lührmann, Reinhard; Wahl, Markus C.

    2010-01-01

    Background The removal of introns from pre-mRNA is carried out by a large macromolecular machine called the spliceosome. The peptidyl-prolyl cis/trans isomerase PPIL1 is a component of the human spliceosome and binds to the spliceosomal SKIP protein via a binding site distinct from its active site. Principal Findings Here, we have studied the PPIL1 protein and its interaction with SKIP biochemically and by X-ray crystallography. A minimal linear binding epitope derived from the SKIP protein could be determined using a peptide array. A 36-residue region of SKIP centred on an eight-residue epitope suffices to bind PPIL1 in pull-down experiments. The crystal structure of PPIL1 in complex with the inhibitor cyclosporine A (CsA) was obtained at a resolution of 1.15 Å and exhibited two bound Cd2+ ions that enabled SAD phasing. PPIL1 residues that have previously been implicated in binding of SKIP are involved in the coordination of Cd2+ ions in the present crystal structure. Employing the present crystal structure, the determined minimal binding epitope and previously published NMR data [1], a molecular docking study was performed. In the docked model of the PPIL1·SKIP interaction, a proline residue of SKIP is buried in a hydrophobic pocket of PPIL1. This hydrophobic contact is encircled by several hydrogen bonds between the SKIP peptide and PPIL1. Conclusion We characterized a short, linear epitope of SKIP that is sufficient to bind the PPIL1 protein. Our data indicate that this SKIP peptide could function in recruiting PPIL1 into the core of the spliceosome. We present a molecular model for the binding mode of SKIP to PPIL1 which emphasizes the versatility of cyclophilin-type PPIases to engage in additional interactions with other proteins apart from active site contacts despite their limited surface area. PMID:20368803

  16. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  17. PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration

    PubMed Central

    Janssens, Veerle; Zwaenepoel, Karen; Rossé, Carine; Petit, Marleen M. R.; Goris, Jozef; Parker, Peter J.

    2017-01-01

    Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn2+-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP–PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130–LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics. PMID:26945059

  18. Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate

    PubMed Central

    Olivieri, Lilian; Gardebien, Fabrice

    2014-01-01

    With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant of this intermediate should be decreased, resulting in an increase of the affinity constant . The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state. PMID:25502559

  19. Arabidopsis acyl-CoA-binding protein ACBP3 participates in plant response to hypoxia by modulating very-long-chain fatty acid metabolism.

    PubMed

    Xie, Li-Juan; Yu, Lu-Jun; Chen, Qin-Fang; Wang, Feng-Zhu; Huang, Li; Xia, Fan-Nv; Zhu, Tian-Ren; Wu, Jian-Xin; Yin, Jian; Liao, Bin; Yao, Nan; Shu, Wensheng; Xiao, Shi

    2015-01-01

    In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by a family of six genes (ACBP1 to ACBP6), and are essential for diverse cellular activities. Recent investigations suggest that the membrane-anchored ACBPs are involved in oxygen sensing by sequestration of group VII ethylene-responsive factors under normoxia. Here, we demonstrate the involvement of Arabidopsis ACBP3 in hypoxic tolerance. ACBP3 transcription was remarkably induced following submergence under both dark (DS) and light (LS) conditions. ACBP3-overexpressors (ACBP3-OEs) showed hypersensitivity to DS, LS and ethanolic stresses, with reduced transcription of hypoxia-responsive genes as well as accumulation of hydrogen peroxide in the rosettes. In contrast, suppression of ACBP3 in ACBP3-KOs enhanced plant tolerance to DS, LS and ethanol treatments. By analyses of double combinations of OE-1 with npr1-5, coi1-2, ein3-1 as well as ctr1-1 mutants, we observed that the attenuated hypoxic tolerance in ACBP3-OEs was dependent on NPR1- and CTR1-mediated signaling pathways. Lipid profiling revealed that both the total amounts and very-long-chain species of phosphatidylserine (C42:2- and C42:3-PS) and glucosylinositolphosphorylceramides (C22:0-, C22:1-, C24:0-, C24:1-, and C26:1-GIPC) were significantly lower in ACBP3-OEs but increased in ACBP3-KOs upon LS exposure. By microscale thermophoresis analysis, the recombinant ACBP3 protein bound VLC acyl-CoA esters with high affinities in vitro. Further, a knockout mutant of MYB30, a master regulator of very-long-chain fatty acid (VLCFA) biosynthesis, exhibited enhanced sensitivities to LS and ethanolic stresses, phenotypes that were ameliorated by ACBP3-RNAi. Taken together, these findings suggest that Arabidopsis ACBP3 participates in plant response to hypoxia by modulating VLCFA metabolism. © 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  20. Retest imaging of [11C]NOP-1A binding to nociceptin/orphanin FQ peptide (NOP) receptors in brain of healthy humans

    PubMed Central

    Lohith, Talakad G.; Zoghbi, Sami S.; Morse, Cheryl L.; Araneta, Maria D. Ferraris; Barth, Vanessa N.; Goebl, Nancy A.; Tauscher, Johannes T.; Pike, Victor W.; Innis, Robert B.; Fujita, Masahiro

    2013-01-01

    [11C]NOP-1A is a novel high-affinity PET ligand for imaging nociceptin/orphanin FQ peptide (NOP) receptors. Here, we report reproducibility and reliability measures of binding parameter estimates for [11C]NOP-1A binding in brain of healthy humans. After intravenous injection of [11C]NOP-1A, PET scans were conducted twice on eleven healthy volunteers on the same (10/11 subjects) or different (1/11 subjects) days. Subjects underwent serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (VT; a measure of receptor density) was determined by compartmental (one- and two-tissue) modeling in large regions and by simpler regression methods (graphical Logan and bilinear MA1) in both large regions and voxel data. Retest variability and intraclass correlation coefficient (ICC) of VT were determined as measures of reproducibility and reliability, respectively. Regional [11C]NOP-1A uptake in brain was high, with a peak radioactivity concentration of 4 – 7 SUV (standardized uptake value) and a rank order of putamen > cingulate cortex > cerebellum. Brain time-activity curves fitted well in 10 of 11 subjects by unconstrained two-tissue compartmental model. The retest variability of VT was moderately good across brain regions except cerebellum, and was similar across different modeling methods, averaging 12% for large regions and 14% for voxel-based methods. The retest reliability of VT was also moderately good in most brain regions, except thalamus and cerebellum, and was similar across different modeling methods averaging 0.46 for large regions and 0.48 for voxels having gray matter probability > 20%. The lowest retest variability and highest retest reliability of VT was achieved by compartmental modeling for large regions, and by the parametric Logan method for voxel-based methods. Moderately good reproducibility and reliability measures of VT for [11C]NOP-1A make it a useful PET ligand for comparing NOP receptor binding

  1. Gender-specific association between the cytoplasmic poly(A) binding protein 4 rs4660293 single nucleotide polymorphism and serum lipid levels.

    PubMed

    Wu, Jian; Yin, Rui-Xing; Guo, Tao; Lin, Quan-Zhen; Shen, Shao-Wen; Sun, Jia-Qi; Shi, Guang-Yuan; Wu, Jin-Zhen; Yang, De-Zhai; Lin, Wei-Xiong

    2015-09-01

    Cytoplasmic poly(A) binding protein 4 (PABPC4) is an RNA-processing protein which has an important role in regulating gene expression. The association of the PABPC4 rs4660293 single nucleotide polymorphism (SNP) and serum lipid profiles has, to the best of our knowledge, not previously been studied in the Chinese population. The present study aimed to investigate the association between the PABPC4 rs4660293 SNP and several environmental factors with serum lipid levels in the Mulao and Han populations. A total of 727 individuals of Mulao nationality and 729 individuals of Han nationality were randomly selected from stratified randomized samples from a previous study by our group. Genotypes of the PABPC4 rs4660293 SNP were determined via polymerase chain reaction and restriction fragment length polymorphism analyses and subsequently confirmed by direct sequencing. Serum levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein (Apo) B were higher in the Mulao group than those in the Han group (P<0.01 for each). The genotypic and allelic frequencies of the PABPC4 rs4660293 SNP were significantly different between males and females in the Mulao population (P<0.05 for each), while no significant difference was detected between those of males and females amongst the Han population. The frequency of the G allele was higher in Mulao males than in Mulao females (22.12 vs. 13.44%). The G allele carriers were found to have higher total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and ApoAI levels in Han females but not in Han males, and lower TC and HDL-C levels in Mulao females but not in Mulao males than those of the G allele non-carriers (P<0.05 for all). These associations were confirmed by multiple linear regression analysis (P<0.05‑0.001). Serum lipid parameters were also correlated with multiple environmental factors (P<0.05‑0.001). The PABPC4 rs4660293 SNP was associated with serum TC, HDL-C, LDL-C and ApoAI levels in these study

  2. Centrally acting hypotensive agents with affinity for 5-HT1A binding sites inhibit forskolin-stimulated adenylate cyclase activity in calf hippocampus.

    PubMed Central

    Schoeffter, P.; Hoyer, D.

    1988-01-01

    (-)- and (+)-enantiomers of pindolol (1 microM and 0.1 mM, respectively). 6. There was an excellent correlation (r = 0.90, P = 0.0001) between the pEC50 values (ranging from 6.4 to 8.7) of the 19 agonists tested at adenylate cyclase and their pKD for 5-HT1A recognition sites. Apparent pKB values of antagonists at adenylate cyclase and their pKD values for 5-HT1A binding sites were also significantly correlated. 7. This study further indicates that the 5-HT1A recognition site and the 5-HT receptor mediating inhibition of adenylate cyclase in hippocampus are the same.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3207999

  3. Neuropsychological Outcomes in Fatty Acid Oxidation Disorders: 85 Cases Detected by Newborn Screening

    ERIC Educational Resources Information Center

    Waisbren, Susan E.; Landau, Yuval; Wilson, Jenna; Vockley, Jerry

    2013-01-01

    Mitochondrial fatty acid oxidation disorders include conditions in which the transport of activated acyl-Coenzyme A (CoA) into the mitochondria or utilization of these substrates is disrupted or blocked. This results in a deficit in the conversion of fat into energy. Most patients with fatty acid oxidation defects are now identified through…

  4. Neuropsychological Outcomes in Fatty Acid Oxidation Disorders: 85 Cases Detected by Newborn Screening

    ERIC Educational Resources Information Center

    Waisbren, Susan E.; Landau, Yuval; Wilson, Jenna; Vockley, Jerry

    2013-01-01

    Mitochondrial fatty acid oxidation disorders include conditions in which the transport of activated acyl-Coenzyme A (CoA) into the mitochondria or utilization of these substrates is disrupted or blocked. This results in a deficit in the conversion of fat into energy. Most patients with fatty acid oxidation defects are now identified through…

  5. The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

    PubMed Central

    Téoulé, François; Brisac, Cynthia; Pelletier, Isabelle; Vidalain, Pierre-Olivier; Jégouic, Sophie; Mirabelli, Carmen; Bessaud, Maël; Combelas, Nicolas; Autret, Arnaud; Tangy, Frédéric; Delpeyroux, Francis

    2013-01-01

    We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. PMID:23926333

  6. Comparative Proteomics Analysis of Phloem Exudates Collected during the Induction of Systemic Acquired Resistance.

    PubMed

    Carella, Philip; Merl-Pham, Juliane; Wilson, Daniel C; Dey, Sanjukta; Hauck, Stefanie M; Vlot, A Corina; Cameron, Robin K

    2016-06-01

    Systemic acquired resistance (SAR) is a plant defense response that provides long-lasting, broad-spectrum pathogen resistance to uninfected systemic leaves following an initial localized infection. In Arabidopsis (Arabidopsis thaliana), local infection with virulent or avirulent strains of Pseudomonas syringae pv tomato generates long-distance SAR signals that travel from locally infected to distant leaves through the phloem to establish SAR In this study, a proteomics approach was used to identify proteins that accumulate in phloem exudates in response to the induction of SAR To accomplish this, phloem exudates collected from mock-inoculated or SAR-induced leaves of wild-type Columbia-0 plants were subjected to label-free quantitative liquid chromatography-tandem mass spectrometry proteomics. Comparing mock- and SAR-induced phloem exudate proteomes, 16 proteins were enriched in phloem exudates collected from SAR-induced plants, while 46 proteins were suppressed. SAR-related proteins THIOREDOXIN h3, ACYL-COENZYME A-BINDING PROTEIN6, and PATHOGENESIS-RELATED1 were enriched in phloem exudates of SAR-induced plants, demonstrating the strength of this approach and suggesting a role for these proteins in the phloem during SAR To identify novel components of SAR, transfer DNA mutants of differentially abundant phloem proteins were assayed for SAR competence. This analysis identified a number of new proteins (m-type thioredoxins, major latex protein-like protein, ULTRAVIOLET-B RESISTANCE8 photoreceptor) that contribute to the SAR response. The Arabidopsis SAR phloem proteome is a valuable resource for understanding SAR long-distance signaling and the dynamic nature of the phloem during plant-pathogen interactions. © 2016 American Society of Plant Biologists. All Rights Reserved.

  7. Overexpression of Arabidopsis ACBP3 enhances NPR1-dependent plant resistance to Pseudomonas syringe pv tomato DC3000.

    PubMed

    Xiao, Shi; Chye, Mee-Len

    2011-08-01

    ACBP3 is one of six Arabidopsis (Arabidopsis thaliana) genes, designated ACBP1 to ACBP6, that encode acyl-coenzyme A (CoA)-binding proteins (ACBPs). These ACBPs bind long-chain acyl-CoA esters and phospholipids and are involved in diverse cellular functions, including acyl-CoA homeostasis, development, and stress tolerance. Recombinant ACBP3 binds polyunsaturated acyl-CoA esters and phospholipids in vitro. Here, we show that ACBP3 plays a role in the plant defense response to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. ACBP3 mRNA was up-regulated upon pathogen infection and treatments using pathogen elicitors and defense-related phytohormones. Transgenic Arabidopsis ACBP3 overexpressors (ACBP3-OEs) showed constitutive expression of pathogenesis-related genes (PR1, PR2, and PR5), cell death, and hydrogen peroxide accumulation in leaves. Consequently, ACBP3-OEs displayed enhanced resistance to the bacterial pathogen P. syringae DC3000. In contrast, the acbp3 T-DNA insertional mutant was more susceptible and exhibited lower PR gene transcript levels upon infection. Using the ACBP3 OE-1 line in combination with nonexpressor of PR genes1 (npr1-5) or coronatine-insensitive1 (coi1-2), we concluded that the enhanced PR gene expression and P. syringae DC3000 resistance in the ACBP3-OEs are dependent on the NPR1-mediated, but not the COI1-mediated, signaling pathway. Given that ACBP3-OEs showed greater susceptibility to infection by the necrotrophic fungus Botrytis cinerea while the acbp3 mutant was less susceptible, we suggest that ACBP3 plays a role in the plant defense response against biotrophic pathogens that is distinct from necrotrophic pathogens. ACBP3 function in plant defense was supported further by bioinformatics data showing up-regulation of many biotic and abiotic stress-related genes in ACBP3 OE-1 in comparison with the wild type.

  8. Comparative Proteomics Analysis of Phloem Exudates Collected during the Induction of Systemic Acquired Resistance1[OPEN

    PubMed Central

    Wilson, Daniel C.; Dey, Sanjukta; Hauck, Stefanie M.; Vlot, A. Corina; Cameron, Robin K.

    2016-01-01

    Systemic acquired resistance (SAR) is a plant defense response that provides long-lasting, broad-spectrum pathogen resistance to uninfected systemic leaves following an initial localized infection. In Arabidopsis (Arabidopsis thaliana), local infection with virulent or avirulent strains of Pseudomonas syringae pv tomato generates long-distance SAR signals that travel from locally infected to distant leaves through the phloem to establish SAR. In this study, a proteomics approach was used to identify proteins that accumulate in phloem exudates in response to the induction of SAR. To accomplish this, phloem exudates collected from mock-inoculated or SAR-induced leaves of wild-type Columbia-0 plants were subjected to label-free quantitative liquid chromatography-tandem mass spectrometry proteomics. Comparing mock- and SAR-induced phloem exudate proteomes, 16 proteins were enriched in phloem exudates collected from SAR-induced plants, while 46 proteins were suppressed. SAR-related proteins THIOREDOXIN h3, ACYL-COENZYME A-BINDING PROTEIN6, and PATHOGENESIS-RELATED1 were enriched in phloem exudates of SAR-induced plants, demonstrating the strength of this approach and suggesting a role for these proteins in the phloem during SAR. To identify novel components of SAR, transfer DNA mutants of differentially abundant phloem proteins were assayed for SAR competence. This analysis identified a number of new proteins (m-type thioredoxins, major latex protein-like protein, ULTRAVIOLET-B RESISTANCE8 photoreceptor) that contribute to the SAR response. The Arabidopsis SAR phloem proteome is a valuable resource for understanding SAR long-distance signaling and the dynamic nature of the phloem during plant-pathogen interactions. PMID:27208255

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

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

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

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  11. Cholesterol transport in steroid biosynthesis: Role of protein-protein interactions and implications in disease states

    PubMed Central

    Rone, Malena B.; Fan, Jinjiang; Papadopoulos, Vassilios

    2009-01-01

    The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-Rlα TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein-protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role. PMID:19286473

  12. Performance characteristics of diffusive gradients in thin films equipped with a binding gel layer containing precipitated ferrihydrite for measuring arsenic(V), selenium(VI), vanadium(V), and antimony(V).

    PubMed

    Luo, Jun; Zhang, Hao; Santner, Jakob; Davison, William

    2010-11-01

    Measurements at high spatial resolution by DGT (diffusive gradients in thin films) require a binding agent that is homogeneously distributed in the binding layer. Formation of ferrihydrite by in situ precipitation within a hydrogel has been previously shown to meet these requirements for the measurement of oxyanions by DGT. Here, we report for the first time detailed performance characteristics of the binding gel and associated DGT devices obtained by deployment in known solutions. To allow comparison of measured and theoretical accumulation of As(V), Se(VI), V(V), and Sb(V), their diffusion coefficients were determined using an independent diffusion cell. Theoretical responses were obtained irrespective of ionic strength (1-100 mmol L(-1)) and pH (3-8), except for Se above pH 7.8 and V below pH 5. Calculated detection limits, based on deployment times of 1 day, were lower than those for devices made with a binding gel cast with a ferrihydrite slurry, and the measured capacity of the binding layer was also superior. There was no evidence for interference from other oxyanions, but binding performance showed some deterioration after 38 days of storage. The potential capability for measuring labile forms of these oxyanions in acidic to neutral, fresh to brackish waters was demonstrated.

  13. A poly(A) binding protein-specific sequence motif: MRTENGKSKGFGFVC binding to mRNA poly(A) and polynucleotides and its role on mRNA translation.

    PubMed

    Rubin, H N; Halim, M N; Leavis, P C

    1994-06-01

    A consensus sequence (GKSKGFGFV) was recognized in all the sequenced poly(A) binding proteins. We synthesized a 15-amino acid peptide (corresponding to 354-368 in the yeast poly(A) binding protein) which includes the consensus sequence to test its binding affinity to different nucleotides, polynucleotides and mRNA with or without a poly(A) tail. Biochemical and biophysical studies revealed that the 15-amino acid peptide has a strong binding affinity to poly(A) alone or poly(A) attached at the 3' end of mRNA. Circular dichroism spectroscopy demonstrated that the secondary structure of the 15-mer is consistent with that expected based on the structure of the native RNP domain. Furthermore, among the various mononucleotides performed in the present studies, ATP was preferentially found to bind to the 15-mer. To further examine the biological significance of the binding of the 15-mer to the poly(A) tail of mRNA, in vitro translation of the mRNA poly(A)+ in the presence of the 15-mer drastically increased globin synthesis by almost 2-fold, while translation of the deadenylated mRNA in the presence of the 15-mer almost did not alter the rate of incorporation of radiolabeled leucine into globin.

  14. The structure of SSO2064, the first representative of Pfam family PF01796, reveals a novel two-domain zinc-ribbon OB-fold architecture with a potential acyl-CoA-binding role

    PubMed Central

    Krishna, S. Sri; Aravind, L.; Bakolitsa, Constantina; Caruthers, Jonathan; Carlton, Dennis; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L.; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2010-01-01

    SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein–protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0). PMID:20944206

  15. Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

    PubMed Central

    Scanu, A M; Miles, L A; Fless, G M; Pfaffinger, D; Eisenbart, J; Jackson, E; Hoover-Plow, J L; Brunck, T; Plow, E F

    1993-01-01

    Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a). Images PMID:8423225

  16. The molecular chaperone TRiC/CCT binds to the Trp-Asp 40 (WD40) repeat protein WDR68 and promotes its folding, protein kinase DYRK1A binding, and nuclear accumulation.

    PubMed

    Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

    2014-11-28

    Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68.

  17. Poly(A)-Binding Protein 1 Partially Relocalizes to the Nucleus during Herpes Simplex Virus Type 1 Infection in an ICP27-Independent Manner and Does Not Inhibit Virus Replication▿

    PubMed Central

    Salaun, C.; MacDonald, A. I.; Larralde, O.; Howard, L.; Lochtie, K.; Burgess, H. M.; Brook, M.; Malik, P.; Gray, N. K.; Graham, S. V.

    2010-01-01

    Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection. PMID:20573819

  18. Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation [corrected and republished article originally printed in J Cell Biol 1988 May;106(5):1723-34

    PubMed Central

    1988-01-01

    Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of approximately 2.3 X 10(-9) cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusion-mediated aggregation. PMID:3170634

  19. The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants.

    PubMed

    Sonnenfield, J M; Burns, C M; Higgins, C F; Hinton, J C

    2001-02-01

    The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.

  20. In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

    SciTech Connect

    Thangima Zannat, Mst.; Bhattacharjee, Rumpa B.; Bag, Jnanankur

    2011-06-03

    Highlights: {yields} PABP knock down and cell apoptosis. {yields} Nuclear translocation of GAPDH in PABP depleted cells. {yields} Role of p53 in apoptosis of PABP depleted cells. {yields} Bax translocation and cytochrome C release and caspase 3 activation following PABP depletion. {yields} Association of p53 with Bcl2 and Bax. -- Abstract: The cytoplasmic poly (A) binding protein (PABP) interacts with 3' poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

  1. The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

    PubMed

    Copsey, Alice C; Cooper, Simon; Parker, Robert; Lineham, Ella; Lapworth, Cuzack; Jallad, Deema; Sweet, Steve; Morley, Simon J

    2017-08-30

    DDX3X, a helicase, can interact directly with mRNA and translation initiation factors, regulating the selective translation of mRNAs that contain a structured 5' untranslated region. This activity modulates the expression of mRNAs controlling cell cycle progression and mRNAs regulating actin dynamics, contributing to cell adhesion and motility. Previously, we have shown that ribosomes and translation initiation factors localise to the leading edge of migrating fibroblasts in loci enriched with actively translating ribosomes, thereby promoting steady-state levels of ArpC2 and Rac1 proteins at the leading edge of cells during spreading. As DDX3X can regulate Rac1 levels, cell motility and metastasis, we have examined DDX3X protein interactions and localisation using many complementary approaches. We now show that DDX3X can physically interact and co-localise with poly(A)-binding protein 1 and caprin-1 at the leading edge of spreading cells. Furthermore, as depletion of DDX3X leads to decreased cell motility, this provides a functional link between DDX3X, caprin-1 and initiation factors at the leading edge of migrating cells to promote cell migration and spreading. © 2017 The Author(s).

  2. La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

    PubMed

    Yang, Ruiqing; Gaidamakov, Sergei A; Xie, Jingwei; Lee, Joowon; Martino, Luigi; Kozlov, Guennadi; Crawford, Amanda K; Russo, Amy N; Conte, Maria R; Gehring, Kalle; Maraia, Richard J

    2011-02-01

    The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.

  3. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    SciTech Connect

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  4. Improved method for analysis of RNA present in long-term preserved thyroid cancer tissue of atomic bomb survivors.

    PubMed

    Hamatani, Kiyohiro; Eguchi, Hidetaka; Mukai, Mayumi; Koyama, Kazuaki; Taga, Masataka; Ito, Reiko; Hayashi, Yuzo; Nakachi, Kei

    2010-01-01

    Since many thyroid cancer tissue samples from atomic bomb (A-bomb) survivors have been preserved for several decades as unbuffered formalin-fixed, paraffin-embedded specimens, molecular oncological analysis of such archival specimens is indispensable for clarifying the mechanisms of thyroid carcinogenesis in A-bomb survivors. Although RET gene rearrangements are the most important targets, it is a difficult task to examine all of the 13 known types of RET gene rearrangements with the use of the limited quantity of RNA that has been extracted from invaluable paraffin-embedded tissue specimens of A-bomb survivors. In this study, we established an improved 5' rapid amplification of cDNA ends (RACE) method using a small amount of RNA extracted from archival thyroid cancer tissue specimens. Three archival thyroid cancer tissue specimens from three different patients were used as in-house controls to determine the conditions for an improved switching mechanism at 5' end of RNA transcript (SMART) RACE method; one tissue specimen with RET/PTC1 rearrangement and one with RET/PTC3 rearrangement were used as positive samples. One other specimen, used as a negative sample, revealed no detectable expression of the RET gene tyrosine kinase domain. We established a 5' RACE method using an amount of RNA as small as 10 ng extracted from long-term preserved, unbuffered formalin-fixed, paraffin-embedded thyroid cancer tissue by application of SMART technology. This improved SMART RACE method not only identified common RET gene rearrangements, but also isolated a clone containing a 93-bp insert of rare RTE/PTC8 in RNA extracted from formalin-fixed, paraffin-embedded thyroid cancer specimens from one A-bomb survivor who had been exposed to a high radiation dose. In addition, in the papillary thyroid cancer of another high-dose A-bomb survivor, this method detected one novel type of RET gene rearrangement whose partner gene is acyl coenzyme A binding domain 5, located on chromosome 10p

  5. Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

    PubMed Central

    Wadum, Majken C T; Villadsen, Jens K; Feddersen, Søren; Møller, Rikke S; Neergaard, Thomas B F; Kragelund, Birthe B; Højrup, Peter; Faergeman, Nils J; Knudsen, Jens

    2002-01-01

    Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters. PMID:12071849

  6. Modelling of vitamin A binding to tRNA.

    PubMed

    N'soukpoé-Kossi, C N; Bourassa, P; Mandeville, J S; Tajmir-Riahi, H A

    2014-10-01

    The binding sites of retinol and retinoic acid with tRNA are located in aqueous solution at physiological conditions using constant tRNA concentration and various retinoid contents. FTIR, CD, fluorescence spectroscopic methods and molecular modelling were used to determine retinoid binding sites, the binding constant and the effects of retinol and retinoic acid complexation on tRNA conformation and aggregation. Structural analysis showed that retinol and retinoic acid bind tRNA via G-C and A-U base pairs with overall binding constants of Kret-tRNA=2.0 (±0.40)×10(4)M(-1) and Kretac-tRNA=6.0 (±1)×10(4)M(-1). The number of binding sites occupied by retinoids on tRNA were 1.4 for retinol-tRNA and 1.7 for retinoic acid-tRNA complexes. Hydrophobic interactions were also observed at high retinol and retinoic acid contents. Molecular modelling showed the participation of several nucleobases in retinoid-tRNA complexation with free binding energy of -4.36 for retinol-tRNA and -4.53kcal/mol for retinoic acid-tRNA adducts.

  7. Lipid A binding proteins in macrophages detected by ligand blotting

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.

  8. Lipid A binding sites in membranes of macrophage tumor cells

    SciTech Connect

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.

    1988-10-15

    Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA, which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.

  9. A Binding Model and Similarity for Flexible Modular Proteins

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Feinauer, Christoph J.; Hofmann, Andreas; Goldt, Sebastian; Liu, Lei; Heermann, Dieter W.

    2013-03-01

    Modular proteins are one of the most commonly found disordered protein motifs. An example is CTCF, a protein that has been named the master waver of the genome i.e., the organizer of the 3D structure of the chromosomes. Using NMR and numerical simulations, much progress has been made in understanding their various functions and ways of binding. Modular proteins are often composed of protein modules interconnected by flexible linkers. They can be imagined as ``beads on a string.'' We argue that when the number of beads is small, these structures behave like a self avoiding random walk. Nevertheless, when binding to a target, linkers can fold in more ordered and stable states. At the same time, folding can influence functional roles. We show that the flexibility of the linkers can boost binding affinity. As a result of flexibility, the conformations of these proteins before and after binding are different. So this implies that generic binding site prediction methods may fail. To deal with this we introduce a new methodology to characterize and compare these flexible structures. Employing topological concepts we propose a method which intrinsically fuses topology and geometry. GM gratefully acknowledges support from the HGS-MathComp and the RTG 1653.

  10. Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

    PubMed Central

    Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2016-01-01

    Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661

  11. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

    Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast. PMID:25265

  12. ACAT-selective and nonselective DGAT1 inhibition: adrenocortical effects--a cross-species comparison.

    PubMed

    Floettmann, Jan Eike; Buckett, Linda K; Turnbull, Andrew V; Smith, Tim; Hallberg, Carina; Birch, Alan; Lees, David; Jones, Huw B

    2013-01-01

    Acyl-coenzyme A: cholesterol O-Acyltransferase (ACAT) and Acyl-coenzyme A: diacylglycerol O-acyltransferase (DGAT) enzymes play important roles in synthesizing neutral lipids, and inhibitors of these enzymes have been investigated as potential treatments for diabetes and other metabolic diseases. Administration of a Acyl-coenzyme A: diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor with very limited cellular selectivity over ACAT resulted in significant adrenocortical degenerative changes in dogs. These changes included macrosteatotic vacuolation associated with adrenocyte cell death in the zonae glomerulosa and fasciculata and minimal to substantial mixed inflammatory cell infiltration and were similar to those described previously for some ACAT inhibitors in dogs. In the mouse, similar but only transient adrenocortical degenerative changes were seen as well as a distinctive nondegenerative reduction in cortical fine vacuolation. In the marmoset, only the distinctive nondegenerative reduction in cortical fine vacuolation was observed, suggesting that the dog, followed by the mouse, is the most sensitive species for cortical degeneration. Biochemical analysis of adrenal cholesterol and cholesteryl ester indicated that the distinctive reduction in cortical fine vacuolation correlated with a significant reduction in cholesteryl ester in the mouse and marmoset, whereas no significant reduction in cholestryl ester, but an increase in free cholesterol was observed in dogs. Administration of a DGAT1 inhibitor with markedly improved selectivity over ACAT to the marmoset and the mouse resulted in no adrenal pathology at exposures sufficient to cause substantial DGAT1 but not ACAT inhibition, thereby implicating ACAT rather than DGAT1 inhibition as the probable cause of the observed adrenal changes. Recognizing that the distinctive nondegenerative reduction in cortical fine vacuolation in the mouse could be used as a histopathological biomarker for an in vivo model of

  13. Biological Activities of 2-Mercaptobenzothiazole Derivatives: A Review

    PubMed Central

    Azam, Mohammed Afzal; Suresh, Bhojraj

    2012-01-01

    2-Mercaptobenzothiazoles are an important class of bioactive and industrially important organic compounds. These compounds are reported for their antimicrobial and antifungal activities, and are subsequently highlighted as a potent mechanism-based inhibitor of several enzymes like acyl coenzyme A cholesterol acyltransferase, monoamine oxidase, heat shock protein 90, cathepsin D, and c-Jun N-terminal kinases. These derivatives are also known to possess antitubercular, anti-inflammatory, antitumor, amoebic, antiparkinsonian, anthelmintic, antihypertensive, antihyperlipidemic, antiulcer, chemoprotective, and selective CCR3 receptor antagonist activity. This present review article focuses on the pharmacological profile of 2-mercaptobenzothiazoles with their potential activities. PMID:23264933

  14. Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

    PubMed Central

    Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

    2001-01-01

    On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

  15. Lipid synthesis in mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and M. tuberculosis.

    PubMed Central

    Norman, E; De Smet, K A; Stoker, N G; Ratledge, C; Wheeler, P R; Dale, J W

    1994-01-01

    The causative agents of leprosy and tuberculosis, Mycobacterium leprae and Mycobacterium tuberculosis, have a lipid-rich cell envelope which contributes to virulence and antibiotic resistance. Acyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis, consists in mycobacteria of two subunits, one of which is biotinylated. Genes from M. leprae and M. tuberculosis encoding a biotinylated protein have been cloned and sequenced. Analysis of the derived protein sequences demonstrated the presence of biotin-binding sites and putative ATP-bicarbonate interactions sites, consistent with the proteins having a biotin carboxylase function as well as their being biotin carrier proteins. Images PMID:7909542

  16. Knockdown of ACAT-1 reduces amyloidogenic processing of APP.

    PubMed

    Huttunen, Henri J; Greco, Christopher; Kovacs, Dora M

    2007-04-17

    Previous studies have shown that acyl-coenzyme A:cholesterol acyl transferase (ACAT), an enzyme that controls cellular equilibrium between free cholesterol and cholesteryl esters, modulates proteolytic processing of APP in cell-based and animal models of Alzheimer's disease. Here we report that ACAT-1 RNAi reduced cellular ACAT-1 protein by approximately 50% and cholesteryl ester levels by 22% while causing a slight increase in the free cholesterol content of ER membranes. This correlated with reduced proteolytic processing of APP and 40% decrease in Abeta secretion. These data show that even a modest decrease in ACAT activity can have robust suppressive effects on Abeta generation.

  17. A complex comprising phosphatidylinositol 4-kinase IIIβ, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex.

    PubMed

    Ishikawa-Sasaki, Kumiko; Sasaki, Jun; Taniguchi, Koki

    2014-06-01

    Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus Kobuvirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication

  18. Riboflavin therapy. Biochemical heterogeneity in two adult lipid storage myopathies.

    PubMed

    Vergani, L; Barile, M; Angelini, C; Burlina, A B; Nijtmans, L; Freda, M P; Brizio, C; Zerbetto, E; Dabbeni-Sala, F

    1999-12-01

    Two unrelated adult males, aged 36 (patient 1) and 25 (patient 2) years, presented with subacute carnitine-deficient lipid storage myopathy that was totally and partly responsive to riboflavin supplementation in the two patients, respectively. Plasma acyl-carnitine and urinary organic acid profiles indicated multiple acyl coenzyme A dehydrogenase deficiency, which was mild in patient 1 and severe in patient 2. The activities of short-chain and medium-chain acyl coenzyme A dehydrogenases in mitochondrial fractions were decreased, especially in patient 2. This was in agreement with Western blotting results. Flavin-dependent complexes I and II were studied by immunoblotting and densitometric quantification of two-dimensional electrophoresis with comparable results. Complex I was present in normal amounts in both patients, whereas complex II was decreased only in the pretherapy muscle of patient 2. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) concentrations in muscle and isolated mitochondria, and the activity of mitochondrial FAD pyrophosphatase, showed that patient 1 had low levels of FAD (46%) and FMN (49%) in mitochondria, with a significant increase (P < 0.01) in mitochondrial FAD pyrophosphatase (273%) compared with controls. Patient 2 had similar low levels of FAD and FMN in both total muscle (FAD and FMN 22% of controls) and mitochondria (FAD 26%; FMN 16%) and normal activity of mitochondrial FAD pyrophosphatase. All of these biochemical parameters were either totally or partly corrected after riboflavin therapy.

  19. The Inflammatory Response in Acyl-CoA Oxidase 1 Deficiency (Pseudoneonatal Adrenoleukodystrophy)

    PubMed Central

    El Hajj, H. I.; Vluggens, A.; Andreoletti, P.; Ragot, K.; Mandard, S.; Kersten, S.; Waterham, H. R.; Lizard, G.; Wanders, R. J. A.; Reddy, J. K.

    2012-01-01

    Among several peroxisomal neurodegenerative disorders, the pseudoneonatal adrenoleukodystrophy (P-NALD) is characterized by the acyl-coenzyme A oxidase 1 (ACOX1) deficiency, which leads to the accumulation of very-long-chain fatty acids (VLCFA) and inflammatory demyelination. However, the components of this inflammatory process in P-NALD remain elusive. In this study, we used transcriptomic profiling and PCR array analyses to explore inflammatory gene expression in patient fibroblasts. Our results show the activation of IL-1 inflammatory pathway accompanied by the increased secretion of two IL-1 target genes, IL-6 and IL-8 cytokines. Human fibroblasts exposed to very-long-chain fatty acids exhibited increased mRNA expression of IL-1α and IL-1β cytokines. Furthermore, expression of IL-6 and IL-8 cytokines in patient fibroblasts was down-regulated by MAPK, p38MAPK, and Jun N-terminal kinase inhibitors. Thus, the absence of acyl-coenzyme A oxidase 1 activity in P-NALD fibroblasts triggers an inflammatory process, in which the IL-1 pathway seems to be central. The use of specific kinase inhibitors may permit the modulation of the enhanced inflammatory status. PMID:22508517

  20. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil

    PubMed Central

    Burgal, Julie; Shockey, Jay; Lu, Chaofu; Dyer, John; Larson, Tony; Graham, Ian; Browse, John

    2010-01-01

    Summary A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process. PMID:18643899

  1. l-Glyceraldehyde 3-Phosphate, a Bactericidal Agent

    PubMed Central

    Tang, Chu-Tay; Engel, Robert; Tropp, Burton E.

    1977-01-01

    At a concentration of 2.5 mM, dl-glyceraldehyde 3-phosphate has a bactericidal effect upon Escherichia coli. The glycerol 3-phosphate transport system is required for the entry of the biologically active l-enantiomer. l-Glyceraldehyde must be phosphorylated by the cell to exert its full effect upon growth. The addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli caused no preferential inhibition of the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although protein accumulation was less affected. Studies with mutant strains ruled out catabolic glycerol 3-phosphate dehydrogenase, anabolic nicotinamide adenine dinucleotide (phosphate):sn-glycerol 3-phosphate oxidoreductase, and fructose 1,6-diphosphate aldolase as the primary sites of action. l-Glyceraldehyde 3-phosphate is a competitive inhibitor of sn-glycerol 3-phosphate in the reactions catalyzed by acyl coenzyme A:sn-glycerol 3-phosphate acyltransferase (Ki of 1.8 mM) and cytidine 5′-diphosphate-diglyceride:sn-glycerol 3-phosphate phosphatidyltransferase (Ki of 2.7 mM). A Km mutant for the former enzyme was susceptible to the inhibitor. l-Glyceraldehyde 3-phosphate does not affect acyl coenzyme A:lysophosphatidate acyltransferase activity. In vivo, phosphatidylethanolamine and phosphatidylglycerol accumulation are inhibited to the same extent by the addition of dl-glyceraldehyde 3-phosphate to a culture of E. coli. PMID:319747

  2. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.

    PubMed

    Chang, C C; Lee, C Y; Chang, E T; Cruz, J C; Levesque, M C; Chang, T Y

    1998-12-25

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.

  3. Effect of VULM 1457, an ACAT inhibitor, on serum lipid levels and on real time red blood cell flow in diabetic and non-diabetic hamsters fed high cholesterol-lipid diet.

    PubMed

    Vojtassáková, E; Syneková, M; Tazká, D; Mátyás, S; Hózová, R; Sadlonová, I; Svec, P

    2007-12-01

    Acyl-coenzyme A: cholesterol O-acyltransferase (ACAT) catalyzes the formation of cholesterol/fatty acyl-coenzyme A esters. Accumulation of cholesterol esters leads to pathological changes connected with atherosclerosis. We have evaluated effects of a newly synthesized ACAT inhibitor, 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM 1457), on serum lipid (cholesterol and triglycerides) levels and velocity of red blood cells (RBC) in non-diabetic and diabetic hamsters fed on high cholesterol-lipid (HCHL) diet during 3 months. The VULM 1457 effects on the paw microcirculation were assessed using capillary microscopy by measuring (RBC) velocity in vivo. Hamsters fed on HCHL diet became hypercholesterolemic with a dramatic increase in serum lipids accompanied with significantly decreased RBC velocity. Diabetic hamsters fed on HCHL diet had further increased serum lipids with reduction of RBC velocity. The VULM 1457 inhibitor lowered cholesterol levels in both non-diabetic and diabetic hamsters fed on HCHL diet. The greater VULM 1457 effect was shown in diabetic hamsters fed on HCHL diet where VULM 1457 expressed hypotriglycerides effects, too. An improved RBC velocity-pronounced effect was observed in diabetic hamsters fed on HCHL diet treated with VULM 1457. These results suggest that the ACAT inhibitor, VULM 1457, is a prospective hypolipidemic and anti-atherogenic drug which treats diabetes.

  4. Multisite Promiscuity in the Processing of Endogenous Substrates By Human Carboxylesterase 1

    SciTech Connect

    Bencharit, S.; Edwards, C.C.; Morton, C.L.; Howard-Williams, E.L.; Kuhn, P.; Potter, P.M.; Redinbo, M.R.; /North Carolina U. /St. Jude Children's Hosp., Memphis /SLAC, SSRL

    2007-01-16

    Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.

  5. Multisite Promiscuity in the Processing of Endogenous Substrates by Human Carboxylesterase 1

    PubMed Central

    Bencharit, Sompop; Edwards, Carol C.; Morton, Christopher L.; Howard-Williams, Escher L.; Kuhn, Peter; Potter, Philip M.; Redinbo, Matthew R.

    2006-01-01

    Human carboxylesterase 1 (hCE1) is a drug- and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-CoenzymeA:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in various complexes with endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically-distinct ligands, these structures reveal that the enzyme contains two additional ligand binding sites and that each site also exhibits relatively non-specific ligand binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions. PMID:16962139

  6. Patatin-related phospholipase pPLAIIIδ increases seed oil content with long-chain fatty acids in Arabidopsis.

    PubMed

    Li, Maoyin; Bahn, Sung Chul; Fan, Chuchuan; Li, Jia; Phan, Tien; Ortiz, Michael; Roth, Mary R; Welti, Ruth; Jaworski, Jan; Wang, Xuemin

    2013-05-01

    The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, β, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.

  7. Antisense suppression of type 1 diacylglycerol acyltransferase adversely affects plant development in Brassica napus.

    PubMed

    Lock, Yee-Ying; Snyder, Crystal L; Zhu, Weiming; Siloto, Rodrigo M P; Weselake, Randall J; Shah, Saleh

    2009-09-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-coenzyme A (CoA) dependent acylation of sn-1,2-diacylglycerol to form triacylglycerol in the terminal step of seed oil formation. Previous work has suggested that the level of DGAT activity may have a substantial effect on the flow of carbon into triacylglycerol, implying that the enzyme may represent a promising target for seed oil modification through biotechnological approaches. In the current study, Brassica napus DH12075 was transformed with an antisense type 1 DGAT construct, resulting in a reduction in DGAT1 gene expression, total DGAT activity and seed oil content. In addition, reduced seed yield and germination rates were observed along with severe developmental abnormalities. These data suggest that in addition to its critical role in seed oil formation, DGAT1 enzyme may also be important for normal seed development in B. napus, although the underlying mechanism(s) remain to be determined. Copyright © Physiologia Plantarum 2009.

  8. Cardiac Lipotoxicity: Molecular Pathways and Therapeutic Implications

    PubMed Central

    Drosatos, Konstantinos; Schulze, P. Christian

    2013-01-01

    Diabetes and obesity are both associated with lipotoxic cardiomyopathy exclusive of coronary artery disease and hypertension. Lipotoxicities have become a public health concern and are responsible for a significant portion of clinical cardiac disease. These abnormalities may be the result of a toxic metabolic shift to more fatty acid and less glucose oxidation with concomitant accumulation of toxic lipids. Lipids can directly alter cellular structures and activate downstream pathways leading to toxicity. Recent data have implicated fatty acids and fatty acyl coenzyme A, diacylglycerol and ceramide in cellular lipotoxicity, which may be caused by apoptosis, defective insulin signaling, endoplasmic reticulum stress, activation of protein kinase C, MAPK activation or modulation of PPARs. PMID:23508767

  9. Peroxisome Proliferator-Activated Receptor Alpha (PPARα), a Key Regulator of Lipid Metabolism in Avians.

    PubMed

    Navidshad, Bahman; Royan, M

    2016-01-01

    Dietary fatty acids have various effects on cellular metabolism, and many of these effects are carried out through the alteration of the gene expression. Fatty acids upregulate or downregulate the expression of different genes by acting both as agonists or antagonists for nuclear hormone receptors. Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. To date, three PPAR subtypes, α, β, and γ, have been recognized. PPARs regulate various target genes with a role in intracellular and extracellular lipid metabolism, mainly those involved in peroxisomal β-oxidation. PPAR controls several genes involved in lipid metabolism, including Δ-5, Δ-6, and Δ-9 desaturases, acyl-coenzyme A oxidase, and carnitine palmitoyltransferase. The activation of PPARα might thus decrease fat deposits indirectly by raising hepatic fatty acid β-oxidation. PPARα is likely part of the regulating system of energy metabolism in peripheral tissues such as adipose tissue and skeletal muscles.

  10. L-carnitine enhances excretion of propionyl coenzyme A as propionylcarnitine in propionic acidemia.

    PubMed

    Roe, C R; Millington, D S; Maltby, D A; Bohan, T P; Hoppel, C L

    1984-06-01

    Treatment with L-carnitine greatly enhanced the formation and excretion of short-chain acylcarnitines in three patients with propionic acidemia and in three normal controls. The use of fast atom bombardment mass spectrometry and linked scanning at constant magnetic (B) to electric (E) field ratio identified the acylcarnitine as propionylcarnitine in patients with propionic acidemia. The normal children excreted mostly acetylcarnitine. Propionic acidemia and other organic acidurias are characterized by the intramitochondrial accumulation of short-chain acyl-Coenzyme A (CoA) compounds. The substrate specificity of the carnitine acetyltransferase enzyme and its steady state nature appears to facilitate elimination of propionyl groups while restoring the acyl-CoA:free CoA ratio in the mitochondrion. We suggest that L-carnitine may be a useful therapeutic approach for elimination of toxic acyl CoA compounds in several of these disorders.

  11. L-carnitine enhances excretion of propionyl coenzyme A as propionylcarnitine in propionic acidemia.

    PubMed Central

    Roe, C R; Millington, D S; Maltby, D A; Bohan, T P; Hoppel, C L

    1984-01-01

    Treatment with L-carnitine greatly enhanced the formation and excretion of short-chain acylcarnitines in three patients with propionic acidemia and in three normal controls. The use of fast atom bombardment mass spectrometry and linked scanning at constant magnetic (B) to electric (E) field ratio identified the acylcarnitine as propionylcarnitine in patients with propionic acidemia. The normal children excreted mostly acetylcarnitine. Propionic acidemia and other organic acidurias are characterized by the intramitochondrial accumulation of short-chain acyl-Coenzyme A (CoA) compounds. The substrate specificity of the carnitine acetyltransferase enzyme and its steady state nature appears to facilitate elimination of propionyl groups while restoring the acyl-CoA:free CoA ratio in the mitochondrion. We suggest that L-carnitine may be a useful therapeutic approach for elimination of toxic acyl CoA compounds in several of these disorders. PMID:6725560

  12. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  13. Thioesterase II of Escherichia coli Plays an Important Role in 3-Hydroxydecanoic Acid Production

    PubMed Central

    Zheng, Zhong; Gong, Qiang; Liu, Tao; Deng, Ying; Chen, Jin-Chun; Chen, Guo-Qiang

    2004-01-01

    3-Hydroxydecanoic acid (3HD) was produced in Escherichia coli by mobilizing (R)-3-hydroxydecanoyl-acyl carrier protein-coenzyme A transacylase (PhaG, encoded by the phaG gene). By employing an isogenic tesB (encoding thioesterase II)-negative knockout E. coli strain, CH01, it was found that the expressions of tesB and phaG can up-regulate each other. In addition, 3HD was synthesized from glucose or fructose by recombinant E. coli harboring phaG and tesB. This study supports the hypothesis that the physiological role of thioesterase II in E. coli is to prevent the abnormal accumulation of intracellular acyl-coenzyme A. PMID:15240249

  14. ACAT inhibition and amyloid beta reduction.

    PubMed

    Bhattacharyya, Raja; Kovacs, Dora M

    2010-08-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Abeta) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Abeta production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Abeta generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Abeta generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD.

  15. Human acyl-CoA:cholesterol acyltransferase (ACAT) and its potential as a target for pharmaceutical intervention against atherosclerosis.

    PubMed

    Chang, Catherine; Dong, Ruhong; Miyazaki, Akira; Sakashita, Naomi; Zhang, Yi; Liu, Jay; Guo, Michael; Li, Bo-Liang; Chang, Ta-Yuan

    2006-03-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty-acyl-coenzyme A. At the single-cell level, ACAT serves as a regulator of intracellular cholesterol homeostasis. In addition, ACAT supplies cholesteryl esters for lipoprotein assembly in the liver and small intestine. Under pathological conditions, the accumulation of cholesteryl esters produced by ACAT in macrophages contributes to foam cell formation, a hallmark of the early stage of atherosclerosis. Several reviews addressing various aspects of ACAT and ACAT inhibitors are available. This review briefly outlines the current knowledge on the biochemical properties of human ACATs, and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventions against atherosclerosis.

  16. Using self-organizing map (SOM) and support vector machine (SVM) for classification of selectivity of ACAT inhibitors.

    PubMed

    Wang, Ling; Wang, Maolin; Yan, Aixia; Dai, Bin

    2013-02-01

    Using a self-organizing map (SOM) and support vector machine, two classification models were built to predict whether a compound is a selective inhibitor toward the two Acyl-coenzyme A: cholesterol acyltransferase (ACAT) isozymes, ACAT-1 and ACAT-2. A dataset of 97 ACAT inhibitors was collected. For each molecule, the global descriptors, 2D and 3D property autocorrelation descriptors and autocorrelation of surface properties were calculated from the program ADRIANA.Code. The prediction accuracies of the models (based on the training/ test set splitting by SOM method) for the test sets are 88.9 % for SOM1, 92.6 % for SVM1 model. In addition, the extended connectivity fingerprints (ECFP_4) for all the molecules were calculated and the structure-activity relationship of selective ACAT inhibitors was summarized, which may help find important structural features of inhibitors relating to the selectivity of ACAT isozymes.

  17. Potential Clinical Implications of the Urotensin II Receptor Antagonists.

    PubMed

    Tsoukas, Philip; Kane, Emilie; Giaid, Adel

    2011-01-01

    Urotensin II (UII) binds to its receptor, UT, playing an important role in the heart, kidneys, pancreas, adrenal gland, and central nervous system. In the vasculature, it acts as a potent endothelium-independent vasoconstrictor and endothelium-dependent vasodilator. In disease states, however, this constriction-dilation equilibrium is disrupted. There is an upregulation of the UII system in heart disease, metabolic syndrome, and kidney failure. The increase in UII release and UT expression suggest that UII system may be implicated in the pathology and pathogenesis of these diseases by causing an increase in acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) activity leading to smooth muscle cell proliferation and foam cell infiltration, insulin resistance (DMII), as well as inflammation, high blood pressure, and plaque formation. Recently, UT antagonists such as SB-611812, palosuran, and most recently a piperazino-isoindolinone based antagonist have been developed in the hope of better understanding the UII system and treating its associated diseases.

  18. Compartmentation of Peroxisomal Enzymes in Algae of the Group of Prasinophyceae 1

    PubMed Central

    Stabenau, H.; Winkler, U.; Säftel, W.

    1989-01-01

    Using the Prasinophycean algae Platymonas, Heteromastix, Pedinomonas, and Pyramimonas, subcellular distribution of the enzymes of glycolate metabolism and β-oxidation pathway have been studied. Glycolate dehydrogenase, hydroxypyruvate reductase, and glutamate-glyoxylate aminotransferase are located in the mitochondria. In addition, the mitochondria of all four species contain acyl-coenzyme A (CoA) dehydrogenase, enoyl-CoA hydratase, and thiolase. In Platymonas, Heteromastix, and Pedinomonas, organelles with the characteristic structure of peroxisomes have been detected which also contain the enzymes acyl-CoA oxidase, enoyl-CoA hydratase and thiolase. However, catalase could not be demonstrated in either the peroxisome-like organelles or in the whole cells. Images Figure 2 PMID:16666839

  19. 10 CFR 851.7 - Requests for a binding interpretive ruling.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 851.7 Energy DEPARTMENT OF ENERGY WORKER SAFETY AND HEALTH PROGRAM General Provisions § 851.7 Requests... rulings with addressing the same or a related issue. (g) Informal conference. DOE may convene an informal... with respect to the person who requested the ruling. (i) Reliance on interpretive ruling. If DOE issues...

  20. 10 CFR 851.7 - Requests for a binding interpretive ruling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 851.7 Energy DEPARTMENT OF ENERGY WORKER SAFETY AND HEALTH PROGRAM General Provisions § 851.7 Requests... rulings with addressing the same or a related issue. (g) Informal conference. DOE may convene an informal... with respect to the person who requested the ruling. (i) Reliance on interpretive ruling. If DOE issues...

  1. 10 CFR 851.7 - Requests for a binding interpretive ruling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 851.7 Energy DEPARTMENT OF ENERGY WORKER SAFETY AND HEALTH PROGRAM General Provisions § 851.7 Requests... rulings with addressing the same or a related issue. (g) Informal conference. DOE may convene an informal... with respect to the person who requested the ruling. (i) Reliance on interpretive ruling. If DOE issues...

  2. 10 CFR 851.7 - Requests for a binding interpretive ruling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 851.7 Energy DEPARTMENT OF ENERGY WORKER SAFETY AND HEALTH PROGRAM General Provisions § 851.7 Requests... rulings with addressing the same or a related issue. (g) Informal conference. DOE may convene an informal... with respect to the person who requested the ruling. (i) Reliance on interpretive ruling. If DOE issues...

  3. 10 CFR 851.7 - Requests for a binding interpretive ruling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 851.7 Energy DEPARTMENT OF ENERGY WORKER SAFETY AND HEALTH PROGRAM General Provisions § 851.7 Requests... rulings with addressing the same or a related issue. (g) Informal conference. DOE may convene an informal... with respect to the person who requested the ruling. (i) Reliance on interpretive ruling. If DOE issues...

  4. The CENP-T/-W complex is a binding partner of the histone chaperone FACT

    PubMed Central

    Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J.; Sullivan, Kevin F.; Almouzni, Geneviève

    2016-01-01

    The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A–H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres. PMID:27284163

  5. Diverse molecular recognition properties of blood group A binding monoclonal antibodies.

    PubMed

    Gildersleeve, Jeffrey C; Wright, Whitney Shea

    2016-05-01

    Information about specificity and affinity is critical for use of carbohydrate-binding antibodies. Herein, we evaluated eight monoclonal antibodies to the blood group A (BG-A) antigen. Antibodies 87-G, 9A, HE-10, HE-24, HE-193, HE-195, T36 and Z2A were profiled on a glycan microarray to assess specificity, relative affinity and the influence of glycan density on recognition. Our studies highlight several noteworthy recognition properties. First, most antibodies bound GalNAcα1-3Gal and the BG-A trisaccharide nearly as well as larger BG-A oligosaccharides. Second, several antibodies only bound the BG-A trisaccharide when displayed on certain glycan chains. These first two points indicate that the carrier glycan chains primarily influence selectivity, rather than binding strength. Third, binding of some antibodies was highly dependent on glycan density, illustrating the importance of glycan presentation for recognition. Fourth, some antibodies recognized the tumor-associated Tn antigen, and one antibody only bound the variant composed of a GalNAc-alpha-linked to a serine residue. Collectively, these results provide new insights into the recognition properties of anti-BG-A antibodies.

  6. FAM20A binds to and regulates FAM20C localization

    PubMed Central

    Ohyama, Yoshio; Lin, Ju-Hsien; Govitvattana, Nattanan; Lin, I-Ping; Venkitapathi, Sundharamani; Alamoudi, Ahmed; Husein, Dina; An, Chunying; Hotta, Hak; Kaku, Masaru; Mochida, Yoshiyuki

    2016-01-01

    Mutations in the Family with sequence similarity (FAM) 20 gene family are associated with mineralized tissue phenotypes in humans. Among these genes, FAM20A mutations are associated with Amelogenesis Imperfecta (AI) with gingival hyperplasia and nephrocalcinosis, while FAM20C mutations cause Raine syndrome, exhibiting bone and craniofacial/dental abnormalities. Although it has been demonstrated that Raine syndrome associated-FAM20C mutants prevented FAM20C kinase activity and secretion, overexpression of the catalytically inactive D478A FAM20C mutant was detected in both cell extracts and the media. This suggests that FAM20C secretion doesn’t require its kinase activity, and that another molecule(s) may control the secretion. In this study, we found that extracellular FAM20C localization was increased when wild-type (WT), but not AI-forms of FAM20A was co-transfected. On the other hand, extracellular FAM20C was absent in the conditioned media of mouse embryonic fibroblasts (MEFs) derived from Fam20a knock-out (KO) mouse, while it was detected in the media from WT MEFs. We also showed that cells with the conditioned media of Fam20a WT MEFs mineralized, but those with the conditioned media of KO MEFs failed to mineralize in vitro. Our data thus demonstrate that FAM20A controls FAM20C localization that may assist in the extracellular function of FAM20C in mineralized tissues. PMID:27292199

  7. Evaluation of polyacrylonitrile (PAN) as a binding polymer for absorbers used to treat liquid radioactive wastes

    SciTech Connect

    Sebesta, F.; John, J.; Motl, A.; Stamberg, K.

    1995-11-01

    The chemical and radiation stability of polyacrylonitrile (PAN) in the form of beads (B-PAN), similar to the beads of composite absorbers, and one selected composite absorber (ammonium molybdophosphate, the active component in PAN binder [AMP-PAN], a prospective candidate for the treatment of acidic wastes) were studied. Aqueous 1M HNO{sub 3} + 1M NaNO{sub 3}, 1M NaOH + 1M NaNO{sub 3}, and 1M NaOH were chosen as simulants of DOE acidic and alkaline wastes. In addition,radiation stability was determined indistilled water. The chemical stability of B-PAN and AMP-PAN beads was tested for a period up to one month of contact with the solution at ambient temperature. The radiation stability of the beads was checked in a radiation dose range 10{sup 3}--10{sup 6} Gy (10{sup 5}--10{sup 8} rads). In acidic solutions the stability of PAN binder was proved not to be limited by either chemical or radiation decomposition. PAN binder may thus be used for preparing composite absorbers for treatment of acid wastes from DOE facilities. The same conclusion is valid for alkaline solutions with pH up to 13. In highly alkaline solutions (concentration of NAOH higher than I M) and in the presence of NaNO{sub 3}, the stability of the tested polyacrylonitrile polymer was sufficient for applications not extending over 10 days. Cross-linking of the polymer caused by ionizing radiation was found to have a positive influence on chemical stability. This effect enables a longer period of applicability of PAN-based composite absorbers. Because of the high sorption rate achievable with PAN-based absorbers, the stability achieved is sufficient for most applications in the DOE complex. The chemical stability of binding polymer may also be further improved by testing another, more suitable type of polymer from the broad family of polyacrylonitrile polymers.

  8. Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study.

    PubMed

    Allard, M; Geoffre, S; Legendre, P; Vincent, J D; Simonnet, G

    1989-10-23

    An in vitro binding assay, using 125I-YLFQPQRFamide, a newly synthetized iodinated analog of FLFQPQRFamide, in which Phe1 (F) has been substituted by a Tyr (Y), was developed to demonstrate and characterize putative binding sites of this brain morphine modulating peptide. This radioligand bound in a time-dependent manner to rat spinal cord membrane preparation. This binding was dose-dependent, saturable and reversible. Both kinetic data and saturation measured at equilibrium lead to the existence of a homogenous population of high affinity binding sites with a Kd value of 0.09-0.1 nM and a maximal capacity Bmax of 14.5 +/- 2 fmol/mg protein. Results of competition experiments show that both FLFQPQRFamide and its analog YLFQPQRFamide had a similar capacity to inhibit the 125I-YLFQPQRFamide binding, suggesting that this radioiodinated analog is a good tool to study binding characteristics of FLFQPQRFamide receptors. The related octadecapeptide AGEGLSSPFWSLAAPQRFamide, another mammalian morphine modulating peptide competes for radioligand binding with similar potency. Our results also show that mu, delta and kappa opiate receptor agonists as well as the antagonist naloxone were not able to affect binding either in presence or in absence of 120 mM NaCl. Together, these data demonstrate that FLFQPQRFamide does not function as an endogenous opiate receptor antagonist and that is capacity to reduce opiate-induced analgesia is supported by specific binding sites.

  9. 6-alkylsalicylates are selective Tip60 inhibitors and target the acetyl-CoA binding site

    PubMed Central

    Ghizzoni, Massimo; Wu, Jiang; Gao, Tielong; Haisma, Hidde J.; Dekker, Frank J.; Zheng, Y. George

    2011-01-01

    Histone acetyltransferases are important enzymes that regulate various cellular functions, such as epigenetic control of DNA transcription. Development of HAT inhibitors with high selectivity and potency will provide powerful mechanistic tools for the elucidation of the biological functions of HATs and may also have pharmacological value for potential new therapies. In this work, analogs of the known HAT inhibitor anacardic acid were synthesized and evaluated for inhibition of HAT activity. Biochemical assays revealed novel anacardic acid analogs that inhibited the human recombinant enzyme Tip60 selectively compared to PCAF and p300. Enzyme kinetics studies demonstrated that inhibition of Tip60 by one such novel anacardic acid derive, 20, was essentially competitive with Ac-CoA and noncompetitive with the histone substrate. In addition, these HAT inhibitors effectively inhibited acetyltransferase activity of nuclear extracts on the histone H3 and H4 at micromolar concentrations. PMID:22100137

  10. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors

    PubMed Central

    Fang, Bin; Mane-Padros, Daniel; Bolotin, Eugene; Jiang, Tao; Sladek, Frances M.

    2012-01-01

    Nuclear receptors (NRs) regulate gene expression by binding specific DNA sequences consisting of AG[G/T]TCA or AGAACA half site motifs in a variety of configurations. However, those motifs/configurations alone do not adequately explain the diversity of NR function in vivo. Here, a systematic examination of DNA binding specificity by protein-binding microarrays (PBMs) of three closely related human NRs—HNF4α, retinoid X receptor alpha (RXRα) and COUPTF2—reveals an HNF4-specific binding motif (H4-SBM), xxxxCAAAGTCCA, as well as a previously unrecognized polarity in the classical DR1 motif (AGGTCAxAGGTCA) for HNF4α, RXRα and COUPTF2 homodimers. ChIP-seq data indicate that the H4-SBM is uniquely bound by HNF4α but not 10 other NRs in vivo, while NRs PXR, FXRα, Rev-Erbα appear to bind adjacent to H4-SBMs. HNF4-specific DNA recognition and transactivation are mediated by residues Asp69 and Arg76 in the DNA-binding domain; this combination of amino acids is unique to HNF4 among all human NRs. Expression profiling and ChIP data predict ∼100 new human HNF4α target genes with an H4-SBM site, including several Co-enzyme A-related genes and genes with links to disease. These results provide important new insights into NR DNA binding. PMID:22383578

  11. Insights into Laccase Engineering from Molecular Simulations: Toward a Binding-Focused Strategy.

    PubMed

    Monza, Emanuele; Lucas, M Fatima; Camarero, Susana; Alejaldre, Lorea C; Martínez, Angel T; Guallar, Victor

    2015-04-16

    Understanding the molecular determinants of enzyme performance is of primary importance for the rational design of ad hoc mutants. A novel approach, which combines efficient conformational sampling and quick reactivity scoring, is used here to shed light on how substrate oxidation was improved during the directed evolution experiment of a fungal laccase (from Pycnoporus cinnabarinus), an industrially relevant class of oxidoreductases. It is found that the enhanced activity of the evolved enzyme is mainly the result of substrate arrangement in the active site, with no important change in the redox potential of the T1 copper. Mutations at the active site shift the binding mode into a more buried substrate position and provide a more favorable electrostatic environment for substrate oxidation. As a consequence, engineering the binding event seems to be a viable way to in silico evolution of oxidoreductases.

  12. Mechanism of immobilized protein A binding to immunoglobulin G on nanosensor array surfaces.

    PubMed

    Nelson, Justin T; Kim, Sojin; Reuel, Nigel F; Salem, Daniel P; Bisker, Gili; Landry, Markita P; Kruss, Sebastian; Barone, Paul W; Kwak, Seonyeong; Strano, Michael S

    2015-08-18

    Protein A is often used for the purification and detection of antibodies such as immunoglobulin G (IgG) because of its quadrivalent domains that bind to the Fc region of these macromolecules. However, the kinetics and thermodynamics of the binding to many sensor surfaces have eluded mechanistic description due to complexities associated with multivalent interactions. In this work, we use a near-infrared (nIR) fluorescent single-walled carbon nanotube sensor array to obtain the kinetics of IgG binding to protein A, immobilized using a chelated Cu(2+)/His-tag chemistry to hydrogel dispersed sensors. A bivalent binding mechanism is able to describe the concentration dependence of the effective dissociation constant, KD,eff, which varies from 100 pM to 1 μM for IgG concentrations from 1 ng mL(-1) to 100 μg mL(-1), respectively. The mechanism is shown to describe the unusual concentration-dependent scaling demonstrated by other sensor platforms in the literature as well, and a comparison is made between resulting parameters. For comparison, we contrast IgG binding with that of human growth hormone (hGH) to its receptor (hGH-R) which displays an invariant dissociation constant at KD = 9 μM. These results should aid in the use of protein A and other recognition elements in a variety of sensor types.

  13. A binding free energy decomposition approach for accurate calculations of the fidelity of DNA polymerases

    PubMed Central

    Rucker, Robert; Oelschlaeger, Peter; Warshel, Arieh

    2010-01-01

    DNA polymerase β (pol β) is a small eukaryotic enzyme with the ability to repair short single-stranded DNA gaps that has found use as a model system for larger replicative DNA polymerases. For all DNA polymerases, the factors determining their catalytic power and fidelity are the interactions between the bases of the base pair, amino acids near the active site, and the two magnesium ions. In this report, we study effects of all three aspects on human pol β transition state (TS) binding free energies by reproducing a consistent set of experimentally determined data for different structures. Our calculations comprise the combination of four different base pairs (incoming pyrimidine nucleotides incorporated opposite both matched and mismatched purines) with four different pol β structures (wild type and three separate mutations of ionized residues to alanine). We decompose the incoming deoxynucleoside 5′-triphosphate-TS, and run separate calculations for the neutral base part and the highly charged triphosphate part, using different dielectric constants in order to account for the specific electrostatic environments. This new approach improves our ability to predict the effect of matched and mismatched base pairing and of mutations in DNA polymerases on fidelity and may be a useful tool in studying the potential of DNA polymerase mutations in the development of cancer. It also supports our point of view with regards to the origin of the structural control of fidelity, allowing for a quantified description of the fidelity of DNA polymerases. PMID:19842163

  14. Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

    PubMed

    Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-03-25

    Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology and designing enzymes with new functional capabilities. This article is protected by copyright. All rights reserved.

  15. Bordetella pertussis FbpA binds both unchelated iron and iron siderophore complexes.

    PubMed

    Banerjee, Sambuddha; Weerasinghe, Aruna J; Parker Siburt, Claire J; Kreulen, R Timothy; Armstrong, Sandra K; Brickman, Timothy J; Lambert, Lisa A; Crumbliss, Alvin L

    2014-06-24

    Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe(3+) and indirectly as a ferric siderophore complex.

  16. OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

    PubMed Central

    Fazelinia, Hossein; Cirino, Patrick C; Maranas, Costas D

    2009-01-01

    One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two-level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed-integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium-binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium-binding centers demonstrated that they all exhibit high affinities for terbium (Kd ∼ 22, 38, and 55 μM) and can selectively bind calcium over magnesium. PMID:19177362

  17. Beyond the detergent effect: a binding site for sodium dodecyl sulfate (SDS) in mammalian apoferritin

    SciTech Connect

    Liu, Renyu Bu, Weiming; Xi, Jin; Mortazavi, Shirin R.; Cheung-Lau, Jasmina C.; Dmochowski, Ivan J.; Loll, Patrick J.

    2012-05-01

    Using X-ray crystallography and isothermal titration calorimetry, we show that sodium dodecyl sulfate (SDS) binds specifically to a pre-formed internal cavity in horse-spleen apoferritin. Although sodium dodecyl sulfate (SDS) is widely used as an anionic detergent, it can also exert specific pharmacological effects that are independent of the surfactant properties of the molecule. However, structural details of how proteins recognize SDS are scarce. Here, it is demonstrated that SDS binds specifically to a naturally occurring four-helix bundle protein: horse apoferritin. The X-ray crystal structure of the apoferritin–SDS complex was determined at a resolution of 1.9 Å and revealed that the SDS binds in an internal cavity that has previously been shown to recognize various general anesthetics. A dissociation constant of 24 ± 9 µM at 293 K was determined by isothermal titration calorimetry. SDS binds in this cavity by bending its alkyl tail into a horseshoe shape; the charged SDS head group lies in the opening of the cavity at the protein surface. This crystal structure provides insights into the protein–SDS interactions that give rise to binding and may prove useful in the design of novel SDS-like ligands for some proteins.

  18. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  19. Lung Tumor Suppressor GPRC5A Binds EGFR and Restrains Its Effector Signaling.

    PubMed

    Zhong, Shuangshuang; Yin, Huijing; Liao, Yueling; Yao, Feng; Li, Qi; Zhang, Jie; Jiao, Huike; Zhao, Yongxu; Xu, Dongliang; Liu, Shuli; Song, Hongyong; Gao, Yong; Liu, Jingyi; Ma, Lina; Pang, Zhi; Yang, Ruixu; Ding, Chengyi; Sun, Beibei; Lin, Xiaofeng; Ye, Xiaofeng; Guo, Wenzheng; Han, Baohui; Zhou, Binhua P; Chin, Y Eugene; Deng, Jiong

    2015-05-01

    GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a(-/-) mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a(-/-) mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a(-/-) MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a(-/-) mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non-small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801-14. ©2015 AACR. ©2015 American Association for Cancer Research.

  20. Carbonation as a binding mechanism for coal/calcium hydroxide pellets

    SciTech Connect

    Rapp, D.M.

    1991-01-01

    Current coal mining and processing procedures produce a significant quanity of fine coal that is difficult to handle and transport. The objective of this work is to determine if these fines can be economically pelletized with calcium hydroxide, a sulfur capturing sorbent, to produce a clean-burning fuel for fluidized-bed combustors or stoker boilers. To harden these pellets, carbonation, which is the reaction of calcium hydroxide with carbon dioxide to produce a cementitious matrix of calcium carbonate, is being investigated. Previous research indicated that carbonation significantly improved compressive strength, impact and attrition resistance and weatherproofed'' pellets formed with sufficient calcium hydroxide (5 to 10% for minus 28 mesh coal fines).

  1. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... the total shipment charges to the individual shipper, so long as it is provided for in your tariff... as a non-binding estimate subject to § 375.405. (7) Once you load a shipment, failure to execute a new binding estimate or a non-binding estimate signifies you have reaffirmed the original...

  2. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.

    PubMed Central

    Sakaguchi, K; Zambrano, N; Baldwin, E T; Shapiro, B A; Erickson, J W; Omichinski, J G; Clore, G M; Gronenborn, A M; Appella, E

    1993-01-01

    The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8506369

  3. Concanavalin A binds to the endoplasmic reticulum and the starch grain surface of root statocytes.

    PubMed

    Schneider, E M; Sievers, A

    1981-07-01

    Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment.

  4. Dual labeling of a binding protein allows for specific fluorescence detection of native protein.

    PubMed

    Karlström, A; Nygren, P A

    2001-08-01

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), respectively. Biosensor analysis of purified doubly labeled protein showed that high-affinity binding to the Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  5. Being a binding site: characterizing residue composition of binding sites on proteins.

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

  6. Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix

    PubMed Central

    1993-01-01

    Leukemia Inhibitory Factor (LIF) interacts with two classes of high affinity binding sites on rat UMR cells cultured in monolayer. One class of binding sites was found to be localized in the extracellular matrix (ECM) after removal of cells from the culture dish. The interaction of LIF with ECM-localized binding sites is not dependent upon either glycosylation of LIF or the presence of extracellular glycosyaminoglycans. Chemical cross-linking studies demonstrate that LIF interacts with a 200-kD cell-associated protein and a 140-kD ECM- localized protein. A 140-kD protein could also be specifically precipitated from solubilised metabolically radiolabeled UMR ECM by antibodies directed against LIF by virtue of its ability to form a stable complex with unlabeled LIF. In addition, soluble LIF associated with this ECM-localized protein is biologically active in terms of inhibition of ES cell differentiation. The properties of ECM-localized 140-kD species are very similar to those of the secreted form of the LIF receptor suggesting that the ECM localization of LIF and LIF signal transduction may be closely coupled. PMID:8335694

  7. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

    PubMed

    Moore, D D; Marks, A R; Buckley, D I; Kapler, G; Payvar, F; Goodman, H M

    1985-02-01

    Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.

  8. Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin

    SciTech Connect

    Le Trong, Isolde; Chu, Vano; Xing, Yi; Lybrand, Terry P.; Stayton, Patrick S.; Stenkamp, Ronald E.

    2013-06-01

    The crystal structures of two circularly permuted streptavidins probe the role of a flexible loop in the tight binding of biotin. Molecular-dynamics calculations for one of the mutants suggests that increased fluctuations in a hydrogen bond between the protein and biotin are associated with cleavage of the binding loop. Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing the biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.

  9. Decreased GABA-A binding on FMZ-PET in succinic semialdehyde dehydrogenase deficiency.

    PubMed

    Pearl, P L; Gibson, K M; Quezado, Z; Dustin, I; Taylor, J; Trzcinski, S; Schreiber, J; Forester, K; Reeves-Tyer, P; Liew, C; Shamim, S; Herscovitch, P; Carson, R; Butman, J; Jakobs, C; Theodore, W

    2009-08-11

    Succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of GABA metabolism characterized by elevated levels of GABA and gamma-hydroxybutyric acid. Clinical findings include intellectual impairment, hypotonia, hyporeflexia, hallucinations, autistic behaviors, and seizures. Autoradiographic labeling and slice electrophysiology studies in the murine model demonstrate use-dependent downregulation of GABA(A) receptors. We studied GABA(A) receptor activity in human SSADH deficiency utilizing [(11)C]-flumazenil (FMZ)-PET. FMZ binding was measured in 7 patients, 10 unaffected parents, and 8 healthy controls. Data analysis was performed using a reference region compartmental model, with time-activity curve from pons as the input function. Relative parametric binding potential (BP(ND)) was derived, with MRI-based pixel by pixel partial volume correction, in regions of interest drawn on coregistered MRI. In amygdala, hippocampus, cerebellar vermis, frontal, parietal, and occipital cortex, patients with SSADH deficiency had significant reductions in FMZ BP(ND) compared to parents and controls. Mean cortical values were 6.96 +/- 0.79 (controls), 6.89 +/- 0.71 (parents), and 4.88 +/- 0.77 (patients) (F ratio 16.1; p < 0.001). There were no differences between controls and parents in any cortical region. Succinic semialdehyde dehydrogenase (SSADH) deficient patients show widespread reduction in BZPR binding on [(11)C]-flumazenil-PET. Our results suggest that high endogenous brain GABA levels in SSADH deficiency downregulate GABA(A)-BZPR binding site availability. This finding suggests a potential mechanism for neurologic dysfunction in a serious neurodevelopmental disorder, and suggests that PET may be useful to translate studies in animal models to human disease.

  10. Decreased GABA-A binding on FMZ-PET in succinic semialdehyde dehydrogenase deficiency

    PubMed Central

    Pearl, P L.; Gibson, K M.; Quezado, Z; Dustin, I; Taylor, J; Trzcinski, S; Schreiber, J; Forester, K; Reeves-Tyer, P; Liew, C; Shamim, S; Herscovitch, P; Carson, R; Butman, J; Jakobs, C; Theodore, W

    2009-01-01

    Objective: Succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of GABA metabolism characterized by elevated levels of GABA and gamma-hydroxybutyric acid. Clinical findings include intellectual impairment, hypotonia, hyporeflexia, hallucinations, autistic behaviors, and seizures. Autoradiographic labeling and slice electrophysiology studies in the murine model demonstrate use-dependent downregulation of GABA(A) receptors. We studied GABA(A) receptor activity in human SSADH deficiency utilizing [11C]-flumazenil (FMZ)-PET. Methods: FMZ binding was measured in 7 patients, 10 unaffected parents, and 8 healthy controls. Data analysis was performed using a reference region compartmental model, with time-activity curve from pons as the input function. Relative parametric binding potential (BPND) was derived, with MRI-based pixel by pixel partial volume correction, in regions of interest drawn on coregistered MRI. Results: In amygdala, hippocampus, cerebellar vermis, frontal, parietal, and occipital cortex, patients with SSADH deficiency had significant reductions in FMZ BPND compared to parents and controls. Mean cortical values were 6.96 ± 0.79 (controls), 6.89 ± 0.71 (parents), and 4.88 ± 0.77 (patients) (F ratio 16.1; p < 0.001). There were no differences between controls and parents in any cortical region. Conclusions: Succinic semialdehyde dehydrogenase (SSADH) deficient patients show widespread reduction in BZPR binding on [11C]-flumazenil-PET. Our results suggest that high endogenous brain GABA levels in SSADH deficiency downregulate GABA(A)-BZPR binding site availability. This finding suggests a potential mechanism for neurologic dysfunction in a serious neurodevelopmental disorder, and suggests that PET may be useful to translate studies in animal models to human disease. GLOSSARY FMZ = flumazenil; MRS = magnetic resonance spectroscopy; PVC = partial volume correction; ROI = region of interest; SPGR = spoiled gradient recall; SSADH = succinic semialdehyde dehydrogenase. PMID:19667317

  11. Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1

    PubMed Central

    Mamonova, Tatyana; Zhang, Qiangmin; Khajeh, Jahan Ali; Bu, Zimei; Bisello, Alessandro; Friedman, Peter A.

    2015-01-01

    Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar KDs. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development. PMID:26070212

  12. Benzene Derivatives Adsorbed to the Ag(111) Surface: A Binding Site Study

    NASA Astrophysics Data System (ADS)

    Miller, Daniel; Simpson, Scott; Tyminska, Nina; Zurek, Eva; Zurek Group Team

    2015-03-01

    Dispersion corrected Density Functional Theory (DFT) calculations were employed to study the interaction of benzenes mono and disubstituted with functional groups encompassing a region of the activated/deacivated spectrum. Benzenes substituted with weak activating or deactivating groups like methyl and fluoro, respectively, do not have a strong site preference for adsorption to the Ag(111) surface. Strong activating (N(CH3)2) and deactivating (NO2) groups, on the other hand, have a distinct site preference. The nitrogen in the former prefers to lie above a silver atom (top site), but in the latter an Hhcp site of the Ag(111) surface is favored. Benzenes derivatized with classic activating groups donate electron density from the highest occupied molecular orbital (HOMO) of the molecule to the surface, and those functionalized with deactivating groups withdraw electron density from the surface into orbitals that are unoccupied in the gas phase. In the case of disubstituted benzenes, the strong activating/deactivating groups control the site preference and other groups assume sites that are, to a large degree, dictated by their positions on the benzene ring. Surface adsorption alters the relative stabilities of the ortho, meta and para positional isomers of disubstituted benzenes.

  13. Peptide synthesis catalyzed by an antibody containing a binding site for variable amino acids.

    PubMed

    Hirschmann, R; Smith, A B; Taylor, C M; Benkovic, P A; Taylor, S D; Yager, K M; Sprengeler, P A; Benkovic, S J

    1994-07-08

    Monoclonal antibodies, induced with a phosphonate diester hapten, catalyzed the coupling of p-nitrophenyl esters of N-acetyl valine, leucine, and phenylalanine with tryptophan amide to form the corresponding dipeptides. All possible stereoisomeric combinations of the ester and amide substrates were coupled at comparable rates. The antibodies did not catalyze the hydrolysis of the dipeptide product nor hydrolysis or racemization of the activated esters. The yields of the dipeptides ranged from 44 to 94 percent. The antibodies were capable of multiple turnovers at rates that exceeded the rate of spontaneous ester hydrolysis. This achievement suggests routes toward creating a small number of antibody catalysts for polypeptide syntheses.

  14. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

  15. The Phosphatidylserine and Phosphatidylethanolamine Receptor CD300a Binds Dengue Virus and Enhances Infection

    PubMed Central

    Carnec, Xavier; Meertens, Laurent; Dejarnac, Ophélie; Perera-Lecoin, Manuel; Hafirassou, Mohamed Lamine; Kitaura, Jiro; Ramdasi, Rasika; Schwartz, Olivier

    2015-01-01

    ABSTRACT Dengue virus (DENV) is the etiological agent of the major human arboviral disease. We previously demonstrated that the TIM and TAM families of phosphatidylserine (PtdSer) receptors involved in the phagocytosis of apoptotic cells mediate DENV entry into target cells. We show here that human CD300a, a recently identified phospholipid receptor, also binds directly DENV particles and enhances viral entry. CD300a facilitates infection of the four DENV serotypes, as well as of other mosquito-borne viruses such as West Nile virus and Chikungunya virus. CD300a acts as an attachment factor that enhances DENV internalization through clathrin-mediated endocytosis. CD300a recognizes predominantly phosphatidylethanolamine (PtdEth) and to a lesser extent PtdSer associated with viral particles. Mutation of residues in the IgV domain critical for phospholipid binding abrogate CD300a-mediated enhancement of DENV infection. Finally, we show that CD300a is expressed at the surface of primary macrophages and anti-CD300a polyclonal antibodies partially inhibited DENV infection of these cells. Overall, these data indicate that CD300a is a novel DENV binding receptor that recognizes PtdEth and PtdSer present on virions and enhance infection. IMPORTANCE Dengue disease, caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease of humans and is a major global health concern. The molecular bases of DENV-host cell interactions during virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. We recently discovered that the TIM and TAM proteins, two receptor families involved in the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, interact with DENV particles-associated PtdSer through a mechanism that mimics the recognition of apoptotic cells and mediate DENV infection. In this study, we show that CD300a, a novel identified phospholipid receptor, mediates DENV infection. CD300a-dependent DENV infection relies on the direct recognition of phosphatidylethanolamine and to a lesser extent PtdSer associated with viral particles. This study provides novel insights into the mechanisms that mediate DENV entry and reinforce the concept that DENV uses an apoptotic mimicry strategy for viral entry. PMID:26468529

  16. Iterative refinement of a binding pocket model: active computational steering of lead optimization.

    PubMed

    Varela, Rocco; Walters, W Patrick; Goldman, Brian B; Jain, Ajay N

    2012-10-25

    Computational approaches for binding affinity prediction are most frequently demonstrated through cross-validation within a series of molecules or through performance shown on a blinded test set. Here, we show how such a system performs in an iterative, temporal lead optimization exercise. A series of gyrase inhibitors with known synthetic order formed the set of molecules that could be selected for "synthesis." Beginning with a small number of molecules, based only on structures and activities, a model was constructed. Compound selection was done computationally, each time making five selections based on confident predictions of high activity and five selections based on a quantitative measure of three-dimensional structural novelty. Compound selection was followed by model refinement using the new data. Iterative computational candidate selection produced rapid improvements in selected compound activity, and incorporation of explicitly novel compounds uncovered much more diverse active inhibitors than strategies lacking active novelty selection.

  17. Relationship of monoamine oxidase A binding to adaptive and maladaptive personality traits.

    PubMed

    Soliman, A; Bagby, R M; Wilson, A A; Miler, L; Clark, M; Rusjan, P; Sacher, J; Houle, S; Meyer, J H

    2011-05-01

    Monoamine oxidase A (MAOA) is an important enzyme that metabolizes monoamines such as serotonin, norepinephrine and dopamine in the brain. In prefrontal cortex, low MAOA binding is associated with aggression and high binding is associated with major depressive disorder (MDD) and also risk for recurrence of depressive episodes. In rodent models, low MAOA levels are associated with increased aggression and fear conditioning, and decreased social and exploratory investigative behaviors. Our objective was to measure MAOA binding in prefrontal cortex and concurrently evaluate a broad range of validated personality traits. We hypothesized that prefrontal MAOA binding would correlate negatively with angry-hostility, a trait related to aggression/anger, and positively with traits intuitively related to adaptive investigative behavior. Participants were aged 19-49 years, healthy and non-smoking. MAOA binding was measured with [11C]harmine positron emission tomography (PET) in prefrontal brain regions and personality traits were measured with the NEO Personality Inventory Revised (NEO PI-R). Prefrontal MAOA binding correlated negatively with angry-hostility (r=-0.515, p=0.001) and positively with deliberation (r=0.514, p=0.001). In a two-factor regression model, these facets explained 38% of variance in prefrontal MAOA binding. A similar relationship was found in prefrontal cortex subregions. We propose a new continuum describing the relationship between personality and MAOA: deliberate/thoughtful contrasting aggressive/impulsive. Additionally, the association between high MAOA binding and greater deliberation may explain why some people have moderately high levels of MAOA, although very high levels occur during MDD. In health, higher MAOA binding is associated with an adaptive personality facet.

  18. Nuclear DNA helicase II/RNA helicase A binds to filamentous actin.

    PubMed

    Zhang, Suisheng; Buder, Katrin; Burkhardt, Carmen; Schlott, Bernhard; Görlach, Matthias; Grosse, Frank

    2002-01-04

    Nuclear DNA helicase II (NDH II), also designated RNA helicase A, is a multifunctional protein involved in transcription, RNA processing, and transport. Here we report that NDH II binds to F-actin. NDH II was partially purified from HeLa nuclear extracts by ion-exchange chromatography on Bio-Rex 70 and DEAE-Sepharose. Upon gel-filtration chromatography on Sepharose 4B, partially purified NDH II resolved into two distinct peaks. The first NDH II peak, corresponding to the void volume of Sepharose 4B, displayed coelution with an abundant 42-kDa protein that was subsequently identified as actin. Several nuclear proteins such as RNA polymerase II, the U5 small nuclear ribonucleoprotein (RNP)-associated WD40 protein, and heterogeneous nuclear RNPs (hnRNPs) copurified with NDH II. However, only hnRNPs A1 and C were found together with NDH II and actin polymers during gel filtration. NDH II and hnRNP C from the HeLa nuclear extract coeluted with F-actin on Sepharose 4B in an RNase-resistant manner, whereas hnRNP A1 was nearly completely removed from F-actin-associated hnRNP complexes following RNA digestion. The association of NDH II and hnRNP C with F-actin was abolished by gelsolin, an F-actin-depolymerizing protein that fragments actin polymers into oligomers or monomers. Furthermore, NDH II co-immunoprecipitated with F-actin and hnRNP C, respectively. In vitro translated NDH II coeluted with F-actin on Sepharose 4B, whereas no coelution with F-actin was observed for in vitro translated hnRNP A1 or C1. Binding to F-actin requires an intact C terminus of NDH II and most likely a native protein conformation. Electron microscopy indicated a close spatial proximity among NDH II, hnRNP C, and F-actin within the HeLa nucleus. These results suggest an important function of NDH II in mediating the attachment of hnRNP-mRPP RNP complexes to the actin nucleoskeleton for RNA processing, transport, or other actin-related processes.

  19. Campylobacter jejuni FlpA binds fibronectin and is required for maximal host cell adherence.

    PubMed

    Konkel, Michael E; Larson, Charles L; Flanagan, Rebecca C

    2010-01-01

    Campylobacter jejuni is one of the most frequent bacterial causes of food-borne gastrointestinal disease in developed countries. Previous work indicates that the binding of C. jejuni to human intestinal cells is crucial for host colonization and disease. Fibronectin (Fn), a major constituent of the extracellular matrix, is a approximately 250-kDa glycoprotein present at regions of cell-to-cell contact in the intestinal epithelium. Fn is composed of three types of repeating units: type I (approximately 45 amino acids), type II (approximately 60 amino acids), and type III (approximately 90 amino acids). The deduced amino acid sequence of C. jejuni flpA (Cj1279c) contains at least three Fn type III domains. Based on the presence of the Fn type III domains, we hypothesized that FlpA contributes to the binding of C. jejuni to human INT 407 epithelial cells and Fn. We assessed the contribution of FlpA in C. jejuni binding to host cells by in vitro adherence assays with a C. jejuni wild-type strain and a C. jejuni flpA mutant and binding of purified FlpA protein to Fn by enzyme-linked immunosorbent assay (ELISA). Adherence assays revealed the binding of the C. jejuni flpA mutant to INT 407 epithelial cells was significantly reduced compared with that for a wild-type strain. In addition, rabbit polyclonal serum generated against FlpA blocked C. jejuni adherence to INT 407 cells in a concentration-dependent manner. Binding of FlpA to Fn was found to be dose dependent and saturable by ELISA, demonstrating the specificity of the interaction. Based on these data, we conclude that FlpA mediates C. jejuni attachment to host epithelial cells via Fn binding.

  20. Assessment of ferula Gummosa gum as a binding agent in tablet formulations.

    PubMed

    Enauyatifard, Reza; Azadbakht, Mohammad; Fadakar, Yousef

    2012-01-01

    Ferula gummosa Boiss. (Apiaceae) is one of the natural plants of Iran. The whole plant, but especially the root, contains the gum resin "galbanum". A study of the comparative effects of galbanum gum and two standard binding agents--polyvinylpyrolidone and acacia--on characteristics of acetaminophen and calcium carbonate compacts was made. The Ferula gummosa gum was extracted and its swelling index was determined. Acetaminophen and calcium carbonate granules were prepared using the wet granulation method and were evaluated for their micromeritics and flow properties, while the compacts were evaluated for mechanical properties using the hardness, tensile strength and friability. The drug release from acetaminophen compacts were assessed using dissolution studies. The dry powder of Ferula gummosa gum resin (galbanum) yielded 14% w/w of gum using distilled water as extraction solvent. The swelling index indicates that galbanum gum swelled to about 190% of initial volume in distilled water. Thus galbanum gum has the ability to hydrate and swells in cold water. The bulk and tapped densities and the interspace porosity (void porosity) percent of the granules prepared with different binders showed significant difference. The hardness and tensile strength of acetaminophen and calcium carbonate compacts containing various binders was of the rank order PVP > acacia > galbanum gum (p < 0.05) and the friability percent was of the reverse order (p < 0.05). The ranking for the dissolution rate of tablets containing the different binders was PVP> galbanum gum > acacia. The results of mechanical properties of acetaminophen and calcium carbonate compacts indicate that galbanum gum could be useful to produce tablets with desired mechanical characteristics for specific purposes, and could be used as an alternative substitute binder in pharmaceutical industries.

  1. Bordetella pertussis FbpA Binds Both Unchelated Iron and Iron Siderophore Complexes

    PubMed Central

    2015-01-01

    Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe3+ and indirectly as a ferric siderophore complex. PMID:24873326

  2. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... integral part of the bill of lading contract. (4) You must clearly indicate upon each binding estimate's... attachment to be made an integral part of the bill of lading contract and have the individual shipper sign... least one year from the date you made the estimate and keep it as an attachment to be made an...

  3. Hepatic triglyceride synthesis and nonalcoholic fatty liver disease.

    PubMed

    Choi, Steve S; Diehl, Anna Mae

    2008-06-01

    Nonalcoholic fatty liver disease is a spectrum of diseases ranging from simple steatosis to cirrhosis. The hallmark of nonalcoholic fatty liver disease is hepatocyte accumulation of triglycerides. We will review the role of triglyceride synthesis in nonalcoholic fatty liver disease progression and summarize recent findings about triglyceride synthesis inhibition and prevention of progressive disease. Attempts to inhibit triglyceride synthesis in animal models have resulted in improvement in hepatic steatosis. Studies in animal models of nonalcoholic fatty liver disease demonstrate that inhibition of acyl-coenzyme A:diacylglycerol acyltransferase, the enzyme that catalyzes the final step in triglyceride synthesis, results in improvement in hepatic steatosis and insulin sensitivity. We recently confirmed that hepatic specific inhibition of acyl-coenzyme A:diacylglycerol acyltransferase with antisense oligonucleotides improves hepatic steatosis in obese, diabetic mice but, unexpectedly, exacerbated injury and fibrosis in that model of progressive nonalcoholic fatty liver disease. When hepatocyte triglyceride synthesis was inhibited, free fatty acids accumulated in the liver, leading to induction of fatty acid oxidizing systems that increased hepatic oxidative stress and liver damage. These findings suggest that the ability to synthesize triglycerides may, in fact, be protective in obesity. Nonalcoholic fatty liver disease is strongly associated with obesity and peripheral insulin resistance. Peripheral insulin resistance increases lipolysis in adipose depots, promoting increased free fatty acid delivery to the liver. In states of energy excess, such as obesity, the latter normally triggers hepatic triglyceride synthesis. When hepatic triglyceride synthesis is unable to accommodate increased hepatocyte free fatty acid accumulation, however, lipotoxicity results. Thus, rather than being hepatotoxic, liver triglyceride accumulation is actually hepato-protective in obese

  4. ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity1[OPEN

    PubMed Central

    Kimberlin, Athen N.; Chen, Ming; Dunn, Teresa M.

    2016-01-01

    Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoid-like proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network. PMID:27506241

  5. Induction of peroxisomes by butyrate-producing probiotics.

    PubMed

    Weng, Huachun; Endo, Kosuke; Li, Jiawei; Kito, Naoko; Iwai, Naoharu

    2015-01-01

    We previously found that peroxisomal biogenesis factor 11a (Pex11a) deficiency is associated with a reduction in peroxisome abundance and impaired fatty acid metabolism in hepatocytes, and results in steatosis. In the present study, we investigated whether butyrate induces Pex11a expression and peroxisome proliferation, and studied its effect on lipid metabolism. C57BL/6 mice fed standard chow or a high-fat diet (HFD) were treated with tributyrin, 4-phelybutyrate acid (4-PBA), or the butyrate-producing probiotics (Clostridium butyricum MIYAIRI 588 [CBM]) plus inulin (dietary fiber), and the body weight, white adipose tissue, serum triglycerides, mRNA expression, and peroxisome abundance were evaluated. Tributyrin or 4-PBA treatment significantly decreased body weight and increased hepatic mRNA expression of peroxisome proliferator-activated receptor-α (PPARα) and Pex11a. In addition, 4-PBA treatment increased peroxisome abundance and the expression of genes involved in peroxisomal fatty acid β-oxidation (acyl-coenzyme A oxidase 1 and hydroxysteroid [17-beta] dehydrogenase 4). CBM and inulin administration reduced adipose tissue mass and serum triglycerides, induced Pex11a, acyl-coenzyme A oxidase 1, and hydroxysteroid (17-beta) dehydrogenase 4 genes, and increased peroxisome abundance in mice fed standard chow or an HFD. In conclusion, elevation of butyrate availability (directly through administration of butyrate or indirectly via administration of butyrate-producing probiotics plus fiber) induces PPARα and Pex11a and the genes involved in peroxisomal fatty acid β-oxidation, increases peroxisome abundance, and improves lipid metabolism. These results may provide a new therapeutic strategy against hyperlipidemia and obesity.

  6. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

    PubMed Central

    Van Hove, J L; Zhang, W; Kahler, S G; Roe, C R; Chen, Y T; Terada, N; Chace, D H; Iafolla, A K; Ding, J H; Millington, D S

    1993-01-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species. PMID:8488845

  7. Development of a method to control the water evaporation of hatching eggs during incubation.

    PubMed

    Ohi, A; Inoue, N; Furuta, H; Sugawara, M; Ohta, Y

    2010-03-01

    Three experiments were conducted to develop methods to control the amount of water loss and to evaluate the metabolic effects of water condition in the White Leghorn breeder eggs during incubation. One hundred twenty, 54, and 90 Julia strain White Leghorn breeder eggs were incubated at 37.8 degrees C, 60% RH in experiments 1, 2, and 3. In experiment 1, eggs were drilled with various bore diameters of 0, 0.5, 1, 2, 3, 4, and 5 mm on the blunt end of the eggshell. In experiment 2, 4 x 4 mm(2) windows were cut into the eggs or the eggs were drilled with 5 holes of bore diameter 2 mm on the blunt end of eggshell. In experiment 3, eggs were drilled with 1, 3, 5, and 7 holes of diameter 2 mm on the blunt end of eggshell. Eggs were treated on d 3 of each experiment and the amount of water loss was recorded on d 19 of incubation. Embryo growth was evaluated in experiments 2 and 3. In addition, the livers of embryos were collected in the 0-, 1-, 3-, and 5-hole treatment groups after weighing eggs to determine 3-hydroxy acyl coenzyme A dehydrogenase activity. In experiment 1, although higher water loss was observed in all windowed eggs than in control, there were no differences in amount of water loss among all bore diameters. Accordingly, that was not successful to control amount of water loss. In experiment 2, higher water loss was observed in drilled eggs at the same levels in windowed eggs as in control. Drilling holes was a more useful treatment to control amount of water loss on incubated eggs than windowing. In experiment 3, amount of water loss increased linearly with increasing number of holes on the blunt end of eggshell. Hepatic 3-hydroxy acyl coenzyme A dehydrogenase activity increased with increasing the number of drilled holes.

  8. A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA

    PubMed Central

    Hernández, M. Luisa; Whitehead, Lynne; He, Zhesi; Gazda, Valeria; Gilday, Alison; Kozhevnikova, Ekaterina; Vaistij, Fabián E.; Larson, Tony R.; Graham, Ian A.

    2012-01-01

    Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the β-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase. PMID:22760209

  9. Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

    PubMed Central

    Kalscheuer, Rainer; Luftmann, Heinrich; Steinbüchel, Alexander

    2004-01-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts. PMID:15574908

  10. Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease.

    PubMed

    Du, Chunyang; Wu, Ming; Liu, Huan; Ren, Yunzhuo; Du, Yunxia; Wu, Haijiang; Wei, Jinying; Liu, Chuxin; Yao, Fang; Wang, Hui; Zhu, Yan; Duan, Huijun; Shi, Yonghong

    2016-10-01

    Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin

  11. Designing a Binding Interface for Control of Cancer Cell Adhesion via 3D Topography and Metabolic Oligosaccharide Engineering

    PubMed Central

    Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J.

    2011-01-01

    This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac5ManNTGc, a thiol-bearing analogue of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bioorthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

  12. Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel*

    PubMed Central

    Picazo-Juárez, Giovanni; Romero-Suárez, Silvina; Nieto-Posadas, Andrés; Llorente, Itzel; Jara-Oseguera, Andrés; Briggs, Margaret; McIntosh, Thomas J.; Simon, Sidney A.; Ladrón-de-Guevara, Ernesto; Islas, León D.; Rosenbaum, Tamara

    2011-01-01

    The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile585 being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content. PMID:21555515

  13. A Characteristic Back Support Structure in the Bisphenol A-Binding Pocket in the Human Nuclear Receptor ERRγ

    PubMed Central

    Liu, Xiaohui; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2014-01-01

    The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR) γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu342-Asn346 and Leu345-Ile349 ridges on the same α-helix 7 (H7), constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT) is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA. PMID:24978476

  14. The Shelterin Component TPP1 Is a Binding Partner and Substrate for the Deubiquitinating Enzyme USP7*

    PubMed Central

    Zemp, Ivo; Lingner, Joachim

    2014-01-01

    The telomeric shelterin component TPP1 has critical functions in telomeric protein complex assembly and telomerase recruitment and regulation. Here we identify USP7 as a novel interacting protein of the oligonucleotide/oligosaccharide-binding fold of TPP1, which was previously known to recruit telomerase to telomeres. We identify amino acids in TPP1 and USP7 that are critical for their interaction and multiple lysines within TPP1 that are oligo-ubiquitinated and deubiquitinated by USP7. Mutational analysis indicated that human TPP1 does not require ubiquitination for telomere association in contrast to previous observations reported for mouse Tpp1. Ubiquitination of human TPP1 also had no detectable effects on known protein interactions of TPP1 with TIN2, POT1, the CTC1-STN1-TEN1 complex, and telomerase. However, the close proximity of USP7 and telomerase binding sites on TPP1 suggest possible cross-talks. In addition, we found that TPP1 is degraded in a proteasome-dependent manner. Prevention of TPP1 ubiquitination prolonged TPP1 half-life ∼2-fold from 45 to 90 min, and remarkably, proteasome inhibition prompted complete stability of TPP1. This indicates that the proteasome destabilizes TPP1 through both direct and indirect pathways possibly involving TPP1-interacting proteins. Altogether, our work identifies novel regulatory circuits that contribute to TPP1 stability and function. PMID:25172512

  15. The shelterin component TPP1 is a binding partner and substrate for the deubiquitinating enzyme USP7.

    PubMed

    Zemp, Ivo; Lingner, Joachim

    2014-10-10

    The telomeric shelterin component TPP1 has critical functions in telomeric protein complex assembly and telomerase recruitment and regulation. Here we identify USP7 as a novel interacting protein of the oligonucleotide/oligosaccharide-binding fold of TPP1, which was previously known to recruit telomerase to telomeres. We identify amino acids in TPP1 and USP7 that are critical for their interaction and multiple lysines within TPP1 that are oligo-ubiquitinated and deubiquitinated by USP7. Mutational analysis indicated that human TPP1 does not require ubiquitination for telomere association in contrast to previous observations reported for mouse Tpp1. Ubiquitination of human TPP1 also had no detectable effects on known protein interactions of TPP1 with TIN2, POT1, the CTC1-STN1-TEN1 complex, and telomerase. However, the close proximity of USP7 and telomerase binding sites on TPP1 suggest possible cross-talks. In addition, we found that TPP1 is degraded in a proteasome-dependent manner. Prevention of TPP1 ubiquitination prolonged TPP1 half-life ∼ 2-fold from 45 to 90 min, and remarkably, proteasome inhibition prompted complete stability of TPP1. This indicates that the proteasome destabilizes TPP1 through both direct and indirect pathways possibly involving TPP1-interacting proteins. Altogether, our work identifies novel regulatory circuits that contribute to TPP1 stability and function. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition.

    PubMed Central

    Zydowsky, L. D.; Etzkorn, F. A.; Chang, H. Y.; Ferguson, S. B.; Stolz, L. A.; Ho, S. I.; Walsh, C. T.

    1992-01-01

    Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin. PMID:1338979

  17. Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation

    PubMed Central

    Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A.; Suh, Mi Chung; Chye, Mee-Len

    2014-01-01

    The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs. PMID:25053648

  18. Lambda Receptor in the Outer Membrane of Escherichia coli as a Binding Protein for Maltodextrins and Starch Polysaccharides

    PubMed Central

    Ferenci, Thomas; Schwentorat, Marina; Ullrich, Susanne; Vilmart, Jeannine

    1980-01-01

    The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane λ receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coli in a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the λ receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as λ receptor is present. (iv) Using the inhibition of λ receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the λ receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (Kd for maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM. PMID:6445892

  19. Identification of a binding motif in the S5 helix that confers cholesterol sensitivity to the TRPV1 ion channel.

    PubMed

    Picazo-Juárez, Giovanni; Romero-Suárez, Silvina; Nieto-Posadas, Andrés; Llorente, Itzel; Jara-Oseguera, Andrés; Briggs, Margaret; McIntosh, Thomas J; Simon, Sidney A; Ladrón-de-Guevara, Ernesto; Islas, León D; Rosenbaum, Tamara

    2011-07-15

    The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile(585) being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.

  20. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  1. Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1.

    PubMed

    Tymecka, Dagmara; Lipiński, Piotr F J; Fedorczyk, Bartłomiej; Puszko, Anna; Wileńska, Beata; Perret, Gerard Y; Misicka, Aleksandra

    2017-08-01

    Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF165 binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF165/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence. Here, we present a structure-activity relationship study of the systematic optimization of amino acid residues in positions 1-3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF165 binding to NRP-1. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Concanavalin A binds to a mannose-containing ligand in the cell wall of some lichen phycobionts.

    PubMed

    Fontaniella, Blanca; Millanes, Ana-María; Vicente, Carlos; Legaz, María-Estrella

    2004-12-01

    Concanavalin A, the lectin from Canavalia ensiformis, develops arginase activity depending on Mn(2+). The cation cannot be substituted by Ca(2+) which, in addition, inhibits Mn(2+)-supported activity. Fluorescein-labeled Concanavalin A is able to bind to the cell wall of algal cells recently isolated from Evernia prunastri and Xanthoria parietina thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which can be isolated by affinity chromatography. Analysis by SDS-PAGE reveals that the ligand is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural lichen lectins, previously identified as a polygalactosylated urease.

  3. Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition.

    PubMed

    Ye, Zi-Wei; Lung, Shiu-Cheung; Hu, Tai-Hua; Chen, Qin-Fang; Suen, Yung-Lee; Wang, Mingfu; Hoffmann-Benning, Susanne; Yeung, Edward; Chye, Mee-Len

    2016-12-01

    Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.

  4. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

    PubMed Central

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-01-01

    Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. PMID:25040887

  5. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism.

    PubMed

    Klingel, U; Miller, C M; North, A K; Stockley, P G; Baumberg, S

    1995-08-21

    In Bacillus subtilis, the AhrC protein represses genes encoding enzymes of arginine biosynthesis and activates those mediating its catabolism. To determine how this repressor also functions as an activator, we attempted to clone catabolic genes by searching for insertions of the Tn917-lacZ transposon that express AhrC-dependent, arginine-inducible beta-galactosidase activity. One such isolate was obtained. The region upstream of lacZ was subcloned in Escherichia coli in such a way that it could be replaced in the B. subtilis chromosome after appropriate manipulation. Analysis of exonuclease III-derived deletions located an AhrC-dependent, arginine-inducible promoter to within a ca. 1.9 kb fragment. The sequence revealed: the 3' end of an ORF homologous to gdh genes encoding glutamate dehydrogenase, with highest homology to the homologue from Clostridium difficile; the 5' end of an ORF homologous to a Saccharomyces cerevisiae gene encoding delta 1-pyrroline 5-carboxylate dehydrogenase (P5CDH), an enzyme of arginine catabolism; and just upstream of the latter, a sequence with homology to known AhrC binding sites in the upstream part of the biosynthetic argCJBD-cpa-F cluster. The same region has also been sequenced by others as part of the B. subtilis genome sequencing project, revealing that the P5CDH gene is the first in a cluster termed rocABC. Restriction fragments containing the putative AhrC-binding sequence, but not those lacking it, showed retarded electrophoretic mobility in the presence of purified AhrC. A 277 bp AhrC-binding fragment also showed anomalous mobility in the absence of AhrC, consistent with its being intrinsically bent. DNAse I footprinting localized AhrC binding to bp -16/-22 to +1 (the transcription startpoint). Such a location for an activator binding site, i.e. overlapping the transcription start, is unusual.

  6. A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

    PubMed Central

    Rausell, Carolina; Martínez-Ramírez, Amparo Consuelo; García-Robles, Inmaculada; Real, María Dolores

    2000-01-01

    The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age. PMID:10742241

  7. Identification of the molecular mechanisms by which the diterpenoid salvinorin A binds to kappa-opioid receptors.

    PubMed

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Stewart, Jeremy; Zjawiony, Jordan K; Vortherms, Timothy A; Sheffler, Douglas J; Roth, Bryan L

    2005-06-21

    Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorumthat selectively and potently activates kappa-opioid receptors (KORs). Salvinorin A is unique in that it is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1-13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.

  8. A binding site outside the canonical PDZ domain determines the specific interaction between Shank and SAPAP and their function

    PubMed Central

    Zeng, Menglong; Shang, Yuan; Guo, Tingfeng; He, Qinghai; Yung, Wing-Ho; Liu, Kai; Zhang, Mingjie

    2016-01-01

    Shank and SAPAP (synapse-associated protein 90/postsynaptic density-95–associated protein) are two highly abundant scaffold proteins that directly interact with each other to regulate excitatory synapse development and plasticity. Mutations of SAPAP, but not other reported Shank PDZ domain binders, share a significant overlap on behavioral abnormalities with the mutations of Shank both in patients and in animal models. The molecular mechanism governing the exquisite specificity of the Shank/SAPAP interaction is not clear, however. Here we report that a sequence preceding the canonical PDZ domain of Shank, together with the elongated PDZ BC loop, form another binding site for a sequence upstream of the SAPAP PDZ-binding motif, leading to a several hundred-fold increase in the affinity of the Shank/SAPAP interaction. We provide evidence that the specific interaction afforded by this newly identified site is required for Shank synaptic targeting and the Shank-induced synaptic activity increase. Our study provides a molecular explanation of how Shank and SAPAP dosage changes due to their gene copy number variations can contribute to different psychiatric disorders. PMID:27185935

  9. Modeling androgen receptor flexibility: a binding mode hypothesis of CYP17 inhibitors/antiandrogens for prostate cancer therapy.

    PubMed

    Gianti, Eleonora; Zauhar, Randy J

    2012-10-22

    Prostate Cancer (PCa), a leading cause of cancer death worldwide (www.cancer.gov), is a complex malignancy where a spectrum of targets leads to a diversity of PCa forms. A widely pursued therapeutic target is the Androgen Receptor (AR). As a Steroid Hormone Receptor, AR serves as activator of transcription upon binding to androgens and plays a central role in the development of PCa. AR is a structurally flexible protein, and conformational plasticity of residues in the binding-pocket is a key to its ability to accommodate ligands from various chemical classes. Besides direct modulation of AR activity by antagonists, inhibition of cytochrome CYP17 (17α-hydroxylase/17,20-lyase), essential in androgen biosynthesis, has widely been considered an effective strategy against PCa. Interestingly, Handratta et al. (2005) discovered new, potent inhibitors of CYP17 (C-17 steroid derivatives) with pure AR antagonistic properties. Although the antiandrogenic activity of their lead compound (VN/124-1) has been experimentally proven both in vitro and in vivo, no structural data are currently available to elucidate the molecular determinants responsible for these desirable dual inhibitory properties. We implemented a Structure-based Drug Design (SBDD) approach to generate a valuable hypothesis as to the binding modes of steroidal CYP17 inhibitors/antiandrogens against the AR. To deal with the plasticity of residues buried in the Ligand Binding Domain (LBD), we developed a flexible-receptor Docking protocol based on Induced-Fit (IFD) methodology (www.schrodinger.com/). Our results constitute an ideal starting point for the rational design of next-generation analogues of CYP17 inhibitors/antiandrogens as well as an attractive tool to suggest novel chemical classes of AR antagonists.

  10. Use of tar pitch as a binding and reductant of BFD waste to produce reactive materials for environmental applications.

    PubMed

    Amorim, Camila C; Leão, Mônica M D; Dutra, Paula R; Tristão, Juliana C; Magalhães, Fabiano; Lago, Rochel M

    2014-08-01

    In this work, a new approach is presented for the modification of the hazardous steel industry waste BFD (Blast Furnace Dust) into a versatile material for application in environmental remediation processes. Tar pitch, another waste, was used to agglomerate the very fine (submicrometric) dust particles to produce a compact and robust pelletized material that under simple thermal treatment produces notably reactive reduced Fe phases. SEM, TG/DTA, Mössbauer, XRD, Raman, BET and elemental analyses indicated that the tar/BFD composite (1:1wt ratio) pellets treated at 400, 600 and 800°C lead to tar decomposition to form a carbon binding coat concomitant with the reduction of the Fe oxides to produce primarily Fe3O4 (magnetite), FeO (wüstite) and Fe(0). Preliminary reactivity studies indicated that these treated composites, especially at 800°C, are active for the reduction of Cr(VI)aq and for the elimination of textile dye via reduction and the Fenton reaction.

  11. A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule.

    PubMed Central

    Dudley, S C; Todt, H; Lipkind, G; Fozzard, H A

    1995-01-01

    We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel. Images FIGURE 4 FIGURE 5 PMID:8580309

  12. Application of poly (ethyleneimine) solution as a binding agent in DGT technique for measurement of heavy metals in water.

    PubMed

    Sui, Dian-Peng; Fan, Hong-Tao; Li, Jing; Li, You; Li, Qiong; Sun, Ting

    2013-09-30

    A 0.050 mol L(-1) solution of poly (ethyleneimine) (PEI), had been used as a novel binding agent of diffusive gradients in thin-films (DGT) technique (PEI-DGT) for measuring the concentrations of labile Cu(2+), Cd(2+) and Pb(2+) in waters. The binding capacities of the PEI-DGT for Cu(2+), Cd(2+) and Pb(2+) were 11.8, 10.2 and 10.6 μmol L(-1), respectively. The performance of PEI-DGT was independence of pH in the range of 4-8 and ionic strength in the range from 1×10(-4) to 0.1 mol L(-1) (as NaNO3). PEI-DGT could measure 104.7±5.2% of the total concentration of Cd(2+) (0.500 mg L(-1)), 95.2±4.3% of the total Cu(2+) (0.500 mg L(-1)) and 99.2±3.4% of the total Pb(2+) (0.500 mg L(-1)) in synthetic solution. Effects of the ligands on the measurement of labile metals were also investigated in synthetic solutions containing the various concentrations of EDTA and humic acid. In EDTA solution, the concentrations of labile metals measured by PEI-DGT showed good agreement with the theoretical concentrations of free metal ions. In humic acid solution, the concentrations of labile metals measured by PEI-DGT decreased with the increase of the concentrations of humic acid. Several DGT devices with various binding agents, including PEI, sodium polyacrylate and poly(4-styrenesulfonate) solution, were used for the measurement of labile fractions of Cu(2+), Cd(2+) and Pb(2+) in the spiked waters and in mine wastewaters. The results showed that the concentrations of labile metal measured by DGT devices with different binding agents could be significantly different, indicating that the labile fractions of metals were dependent on the binding strength of the binding agents with metals. By choosing binding agents, the useful information on the speciation and bioavailability of the analytes can be provided. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Cyclophilin A Binds to the Viral RNA and Replication Proteins, Resulting in Inhibition of Tombusviral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay

    2013-01-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication. PMID:24089553

  14. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    PubMed

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  15. Carbonation as a binding mechanism for coal/calcium hydroxide pellets. [Quarterly] technical report, March 1, 1993--May 31, 1993

    SciTech Connect

    Lytle, J.; Hackley, K.; Dagamac, M.; Berger, R.; Schanche, G.

    1993-09-01

    This research is an investigation of calcium hydroxide, a sulfur-capturing sorbent, as a binder for coal fines. The reaction of carbon dioxide with calcium hydroxide, referred to as carbonation, is being studied as a method of improving pellet quality. Carbonation forms a cementitious matrix of calcium carbonate. The effect of particle size and compaction pressure on pellet strength was studied using a laboratory hydraulic press. Particle distributions with mean sizes of 200, 90 and 40 microns were tested. The results indicate that pellet strength increased with decreasing particle size and increasing compaction pressure when calcium hydroxide was used as a binder. Pellets containing 10 wt% calcium hydroxide increased in strength by approximately 40% when air dried for one day. This increase in strength is attributed to carbonation of the calcium hydroxide via atmospheric carbon dioxide. Corn starch, an adhesive binder, was tested at the finest particle size. Pellet strength did not increase as a function of increasing compaction pressure. At the finest particle size and highest compaction pressure (18,750 psi), dried pellets formed with 2 wt% corn starch were equivalent in strength to pellets containing 5 wt% calcium hydroxide.

  16. (1) Request for a binding decision on whether Ferdinanda (Compositae) and Ferdinandea (Rubiaceae) are sufficiently alike to be confused

    USDA-ARS?s Scientific Manuscript database

    The genus Ferdinandusa is an attractive, widespread shrub in tropical America, a member of the family Rubiaceae, and is sometimes cultivated for its bright showy flowers. It is a later name for the genus Ferdinandea, which has been confused with the genus Ferdinanda. By officially establishing that ...

  17. Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans

    PubMed Central

    Schons-Fonseca, Luciane; da Silva, Josefa B.; Milanez, Juliana S.; Domingos, Renan H.; Smith, Janet L.; Nakaya, Helder I.; Grossman, Alan D.; Ho, Paulo L.; da Costa, Renata MA

    2016-01-01

    We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. PMID:26762976

  18. Carbonation as a binding mechanism for coal/calcium hydroxide pellets. Technical report, December 1, 1992--February 28, 1993

    SciTech Connect

    Rapp, D.; Lytle, J.; Hackley, K.; Dagamac, M.; Berger, R.; Schanche, G.

    1993-05-01

    Pelletization of fine coal with calcium hydroxide, a sulfur capturing sorbent, represents a method to produce a fuel which will burn in compliance with the recently passed Clean Air Act Amendments (CAAA`s). To harden the pellets, the reaction of carbon dioxide with calcium hydroxide, referred to as carbonation, is being studied. Carbonation forms a bonding matrix of calcium carbonate. This is a two-year research program. This report covers the second quarter of the second year. Research is indicating that 5 to 10 wt% calcium hydroxide pellets can be produced via a roller-and-die pellet mill and air cured to achieve sufficient quality for handling and transportation. This quarter, 1/2 inch-diameter pellets containing 10% calcium hydroxide were demonstrated to gradually react with atmospheric carbon dioxide (3 days) while air drying to achieve compressive strengths equivalent to those attained for fully dried pellets which had been carbonated for one-hour with 100% commercial grade carbon dioxide. It was also demonstrated that an organic, adhesive binder, corn starch, can be very effective at producing strong pellets but drying is required before appreciable pellet strength is attained. For pellets containing 2 wt% corn starch, it was determined that less than 50% of the ultimate strength was achieved as the pellets were dried from 20 wt% to 5 wt% moisture. Strength improved considerably as the pellet moisture content was reduced below 5 wt%.

  19. Carbonation as a binding mechanism for coal/calcium hydroxide pellets. Technical report, September 1, 1991--November 30, 1991

    SciTech Connect

    Rapp, D.M.

    1991-12-31

    Current coal mining and processing procedures produce a significant quanity of fine coal that is difficult to handle and transport. The objective of this work is to determine if these fines can be economically pelletized with calcium hydroxide, a sulfur capturing sorbent, to produce a clean-burning fuel for fluidized-bed combustors or stoker boilers. To harden these pellets, carbonation, which is the reaction of calcium hydroxide with carbon dioxide to produce a cementitious matrix of calcium carbonate, is being investigated. Previous research indicated that carbonation significantly improved compressive strength, impact and attrition resistance and ``weatherproofed`` pellets formed with sufficient calcium hydroxide (5 to 10% for minus 28 mesh coal fines).

  20. Elevated monoamine oxidase a binding during major depressive episodes is associated with greater severity and reversed neurovegetative symptoms.

    PubMed

    Chiuccariello, Lina; Houle, Sylvain; Miler, Laura; Cooke, Robert G; Rusjan, Pablo M; Rajkowska, Grazyna; Levitan, Robert D; Kish, Stephen J; Kolla, Nathan J; Ou, Xiaoming; Wilson, Alan A; Meyer, Jeffrey H

    2014-03-01

    Inadequate treatment response occurs in approximately 40% of major depressive episodes (MDEs), and one approach to solve this is careful matching of treatment to the specific pathologies of MDE. One such biological abnormality is elevated monoamine oxidase A (MAO-A) levels, which occurs in the prefrontal and anterior cingulate cortex (PFC and ACC) during MDE; however, the subtypes for which this abnormality is most prominent are unknown. We hypothesized that MAO-A levels in the PFC and ACC are most elevated in MDE with greater severity and reversed neurovegetative symptoms (hypersomnia and either hyperphagia or weight gain). MAO-A VT (an index of MAO-A density) was measured using [(11)C]harmine positron emission tomography (PET) in 42 subjects with MDEs secondary to major depressive disorder and 37 healthy controls. The effect of severity and reversed neurovegetative symptoms on MAO-A VT in the PFC and ACC was analyzed using a multivariate analysis of variance (MANOVA). Greater severity and reversed neurovegetative symptoms were associated with elevated MAO-A VT in the PFC and ACC (MANOVA, severity: F(2,38)=5.44, p=0.008; reversed neurovegetative symptoms: F(2,38)=5.13, p=0.01). Increased MAO-A level, when greater severity and reversed neurovegetative symptoms are present, may explain the association of these clinical features with a preferential response to MAO inhibitors, which is especially well-evidenced for reversed neurovegetative symptoms in MDE. As MAO-A creates oxidative stress, facilitates apoptosis, and metabolizes monoamines, therapeutics opposing these processes are predicted to best treat MDE with greater severity and reversed neurovegetative symptoms.

  1. Live detection and purification of cells based on the expression of a histone chaperone, HIRA, using a binding peptide

    PubMed Central

    Kochurani, K. J.; Suganya, Annie A.; Nair, Madhumathy G.; Louis, Jiss Maria; Majumder, Aditi; Kumar K., Santhosh; Abraham, Parvin; Dutta, Debasree; Maliekal, Tessy T.

    2015-01-01

    Flowcytometry is a reliable method for identification and purification of live cells from a heterogeneous population. Since permeabilized cells cannot be sorted live in a FACS sorter, its application in isolation of functional cells largely depends on antibodies for surface markers. In various fields of biology we find intracellular markers that reveal subpopulations of biological significance. Cell cycle stage specific molecules, metastatic signature molecules, stemness associated proteins etc. are examples of potential markers that could improve the research and therapy enormously. Currently their use is restricted by lack of techniques that allow live detection. Even though a few methods like aptamers, droplet-based microfluidics and smartflares are reported, their application is limited. Here, for the first time we report a simple, cost-effective and efficient method of live sorting of cells based on the expression of an intracellular marker using a fluorophore-tagged binding peptide. The target molecule selected was a histone chaperone, HIRA, the expression of which can predict the fate of differentiating myoblast. Our results confirm that the peptide shows specific interaction with its target; and it can be used to separate cells with differential expression of HIRA. Further, this method offers high purity and viability for the isolated cells. PMID:26596463

  2. Exploring the capabilities of TDDFT calculations to explain the induced chirality upon a binding process: A simple case, 3-carboxycoumarin

    NASA Astrophysics Data System (ADS)

    Varlan, Aurica; Hillebrand, Mihaela

    2013-03-01

    The induced circular dichroism (ICD) spectra of 3-carboxycoumarin recorded at pH 7.4 in the presence of human and bovine serum albumins were used in correlation with theoretical (TDDFT) calculations to obtain the binding constants and information on the conformational changes of the ligand in the binding site. As it was shown that for the carboxylic acids or the carboxylate ions, the asymmetry element correlated with the occurrence of the ICD band in the presence of proteins is the torsion (τ) of the COOH (COO-) group in respect with the planar π system, TDDFT calculations were performed considering all the geometries characterized by 0 ⩽ |τ| ⩽ 90 deg. The simulated circular dichroism spectrum shows that the sequence of the signs and positions of the bands are correctly predicted as compared to the experimental ICD spectrum for a torsion of the carboxylate group in the range of 60-70 deg.

  3. A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens

    PubMed Central

    Koh, Cho Yeow; Kallur Siddaramaiah, Latha; Ranade, Ranae M.; Nguyen, Jasmine; Jian, Tengyue; Zhang, Zhongsheng; Gillespie, J. Robert; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Fan, Erkang; Hol, Wim G. J.

    2015-01-01

    American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallo­graphically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS. PMID:26249349

  4. High-Temperature Cross-Linking of Carbon Nanotube Multi-Yarn Using Polyvinylpyrrolidone as a Binding Agent.

    PubMed

    Misak, H; Asmatulu, R; Whitman, J; Mall, S

    2015-03-01

    Carbon nanotube (CNT) multi-yarn was cross-linked together at elevated temperatures using a poly- mer, with the intent of improving their strength and electrical conductivity. They were functionalized using an acid treatment and immersed in a bath of different concentrations (0.5%, 0.1%, and 0.2%) of polyvinylpyrrolidone (PVP). Then they were placed in an oven at various temperatures (180 °C, 200 °C, and 220 °C) in order to cause cross-linking among the carbon nanotube yarns. The phys- ical, chemical, electrical, and mechanical properties of the cross-linked yarns were investigated. The yarns cross-linked at higher temperatures and greater concentrations of PVP had a greater increase in linear mass density, indicating that the cross-linking process had worked as expected. Yarns that were cross-linked at lower temperatures had greater tensile strength and better spe- cific electrical conductivity. Those that were treated with a greater concentration of polymer had a greater ultimate tensile strength. All these results are encouraging first step, but still need further development if CNT yarn is to replace copper wire.

  5. Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing.

    PubMed

    Banerjee, Ayan; Vest, Katherine E; Pavlath, Grace K; Corbett, Anita H

    2017-09-07

    The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle. We have used co-immunoprecipitation and mass spectrometry to identify proteins that interact with PABPN1 in mouse skeletal muscles. Among the interacting proteins we identified Matrin 3 (MATR3) as a novel protein interactor of PABPN1. The MATR3 gene is mutated in a form of distal myopathy and amyotrophic lateral sclerosis (ALS). We demonstrate, that like PABPN1, MATR3 is critical for myogenesis. Furthermore, MATR3 controls critical aspects of RNA processing including alternative polyadenylation and intron retention. We provide evidence that MATR3 also binds and regulates the levels of long non-coding RNA (lncRNA) Neat1 and together with PABPN1 is required for normal paraspeckle function. We demonstrate that PABPN1 and MATR3 are required for paraspeckles, as well as for adenosine to inosine (A to I) RNA editing of Ctn RNA in muscle cells. We provide a functional link between PABPN1 and MATR3 through regulation of a common lncRNA target with downstream impact on paraspeckle morphology and function. We extend our analysis to a mouse model of OPMD and demonstrate altered paraspeckle morphology in the presence of endogenous levels of alanine-expanded PABPN1. In this study, we report protein-binding partners of PABPN1, which could provide insight into novel functions of PABPN1 in skeletal muscle and identify proteins that could be sequestered with alanine-expanded PABPN1 in the nuclear aggregates found in OPMD. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein.

    PubMed

    Shepherd, Mark; Heath, Mathew D; Poole, Robert K

    2007-05-01

    NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.

  7. IgG rheumatoid factors and staphylococcal protein A bind to a common molecular site on IgG.

    PubMed

    Nardella, F A; Teller, D C; Barber, C V; Mannik, M

    1985-12-01

    The antigenic determinant on the Fc region of human IgG for two IgG rheumatoid factors (IgG-RF) from patients with rheumatoid arthritis were investigated in detail. The RF did not interact with IgG fragments that contained the C gamma 2 or C gamma 3 region alone, but required the presence of both regions for binding. The RF binding to solid-phase IgG were poorly inhibited by the IgG3 subclass and strongly inhibited by staphylococcal protein A (SPA) (42 kD), and fragment D of SPA (7 kD), indicating that the binding site is most likely the same as the Ga antigenic determinant described for IgM-RF, and is in the same location as the site on IgG that binds SPA. pH titration studies of the RF binding to IgG indicated the involvement of histidine and lysine or tyrosine side chains. Chemical modification studies showed the histidines were involved on the Fc side of the interactions, and tyrosines were involved on both the antigenic and antibody sides of the interactions. Lysines were not involved. The above information, and the knowledge of the number and position in space of the amino acid residues involved in the C gamma 2-C gamma 3 interface region of IgG, the binding site for SPA, and the amino acid substitutions in IgG3 that account for its inability to bind protein A, allowed the identification of the site on IgG that bind IgG-RF. This binding site involves some of the same amino acid side chains, His 435, Tyr 436, and one or both His 433 and 310, and is in the same location as the site that binds SPA. The same site is likely to be a common antigenic determinant for other RF. Furthermore, the described molecular mimicry suggests a biological relationship between bacterial Fc-binding proteins and the production of RF in rheumatoid arthritis.

  8. A characteristic back support structure in the bisphenol A-binding pocket in the human nuclear receptor ERRγ.

    PubMed

    Liu, Xiaohui; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2014-01-01

    The endocrine disruptor bisphenol A (BPA) affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR) γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu342-Asn346 and Leu345-Ile349 ridges on the same α-helix 7 (H7), constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT) is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA.

  9. Cse1p-binding dynamics reveal a binding pattern for FG-repeat nucleoporins on transport receptors.

    PubMed

    Isgro, Timothy A; Schulten, Klaus

    2007-08-01

    Nuclear pore proteins with phenylalanine-glycine repeats are vital to the functional transport of molecules across the nuclear pore complex. The current study investigates the binding of these FG-nucleoporins to the Cse1p:Kap60p:RanGTP nuclear export complex. Fourteen binding spots for FG-nucleoporin peptides are revealed on the surface of Cse1p, and 5 are revealed on the Kap60p surface. Taken together, and along with binding data for two other transport receptors, the data suggest that the ability to bind FG-nucleoporins by itself is not enough to ensure viable nuclear transport. Rather, it is proposed that the density of binding spots on the transport receptor surface is key in determining transport viability. The number of binding spots on the transport receptor surface should be large enough to ensure multiple, simultaneous FG-repeat binding, and their arrangement should be close enough to ensure multiple binding from the same FG-nucleoporin.

  10. Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition.

    PubMed

    Wagh, Dhananjay; Pothineni, Venkata Raveendra; Inayathullah, Mohammed; Liu, Song; Kim, Kwang-Min; Rajadas, Jayakumar

    2015-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 μg/mL (250 μM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia.

  11. Presence of autoantibodies to peptidyl-prolyl cis-trans isomerase (cyclosporin A-binding protein) in systemic lupus erythematosus.

    PubMed

    Harigai, M; Hara, M; Takahashi, N; Kitani, A; Hirose, T; Suzuki, K; Kawakami, M; Hidaka, T; Kawaguchi, Y; Ishizuka, T

    1992-04-01

    Several autoantibodies against cytoplasmic or nuclear components of cells have been reported in autoimmune diseases. We report here a previously unrecognized autoantibody to peptidyl-prolyl cis-trans isomerase (PPIase) in patients with systemic lupus erythematosus (SLE). PPIase, which catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, has recently been found to be identical to cyclophilin, a specific binding protein of a potent immunosuppressant, cyclosporin A. IgG and IgM anti-PPIase antibodies were detected in 40 and 20% of unselected patients with SLE, respectively, by ELISA. The reactivity of these sera was confirmed by immunoblotting experiments. Sera from rheumatoid arthritis patients showed no reactivity and 1 of 8 sera from systemic sclerosis patients and 1 of 25 sera from normal controls showed only weak reactivity. Unexpectedly, the anti-PPIase antibody was unable to inhibit PPIase activity, indicating that the autoantibody recognizes an epitope of PPIase which is different from the active site of PPIase. The levels of the anti-PPIase antibody in SLE patients correlated with remissions and flares of the disease. The anti-PPIase antibody was higher in patients with active SLE than those with inactive disease. The prevalence of the active stage of the disease was significantly higher in IgG anti-PPIase antibody-positive SLE patients as compared to antibody-negative SLE patients. These data define the presence of a new autoantibody against PPIase and its association with the activity and certain clinical manifestations in SLE.

  12. Characterization of Xanthomonas axonopodis pv. citri LexA: recognition of the LexA binding site.

    PubMed

    Yang, M-K; Yang, Y-C; Hsu, C-H

    2002-12-01

    Levels of l exA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri ( X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA protein to the sequence upstream of the lexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA protein binds to the region located between the transcription start site and the translation initiation codon of the lexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN(11)CTAA), located in this region as the binding sequence for the LexA protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.

  13. Repellent taxis in response to nickel ion requires neither Ni2+ transport nor the periplasmic NikA binding protein.

    PubMed

    Englert, Derek L; Adase, Christopher A; Jayaraman, Arul; Manson, Michael D

    2010-05-01

    Ni(2+) and Co(2+) are sensed as repellents by the Escherichia coli Tar chemoreceptor. The periplasmic Ni(2+) binding protein, NikA, has been suggested to sense Ni(2+). We show here that neither NikA nor the membrane-bound NikB and NikC proteins of the Ni(2+) transport system are required for repellent taxis in response to Ni(2+).

  14. Borreliacidal activity of Borrelia metal transporter A (BmtA) binding small molecules by manganese transport inhibition

    PubMed Central

    Wagh, Dhananjay; Pothineni, Venkata Raveendra; Inayathullah, Mohammed; Liu, Song; Kim, Kwang-Min; Rajadas, Jayakumar

    2015-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 μg/mL (250 μM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia. PMID:25709405

  15. Characterization of [3H]-CGP54626A binding to heterodimeric GABAB receptors stably expressed in mammalian cells

    PubMed Central

    Green, Andrew; Walls, Steven; Wise, Alan; Green, Richard H; Martin, Amanda K; Marshall, Fiona H

    2000-01-01

    Functional human GABAB(1a,2) and GABAB(1b,2) receptors have been stably expressed in mammalian CHO K1 cells.Detailed characterization of GABAB ligand binding at each of the receptors has been compared using [3H]-CGP54626A. In cell membranes fractions, [3H]-CGP54626A bound to a single site with a KD of 1.51±1.12 nM, Bmax of 2.02±0.17 pmoles mg protein−1 and 0.86±0.20 nM, Bmax of 5.19±0.57 pmoles mg protein−1 for GABAB(1a,2) and GABAB(1b,2) respectively.In competition binding assays the rank order was identical for both GABAB receptors. For known GABAB agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABAB antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 ⩾2-OH Saclofen ⩾ABPA.The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl2 from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABAB receptors.A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABAB receptors. In competition assays, the rank order was the same for both GABAB(1a,2) and GABAB(1b,2) and consistent with that seen with cell membrane fractions.These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABAB receptor are pharmacologically indistinguishable. PMID:11139457

  16. A binding hotspot in Trypanosoma cruzi histidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens.

    PubMed

    Koh, Cho Yeow; Siddaramaiah, Latha Kallur; Ranade, Ranae M; Nguyen, Jasmine; Jian, Tengyue; Zhang, Zhongsheng; Gillespie, J Robert; Buckner, Frederick S; Verlinde, Christophe L M J; Fan, Erkang; Hol, Wim G J

    2015-08-01

    American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

  17. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  18. Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal specific tubulin

    PubMed Central

    Avdoshina, Valeria; Taraballi, Francesca; Dedoni, Simona; Corbo, Claudia; Paige, Mikell; Kont, Yasemin Saygideğer; Üren, Aykut; Tasciotti, Ennio; Mocchetti, Italo

    2016-01-01

    Human immunodeficiency virus-1 (HIV) promotes synaptic simplification and neuronal apoptosis, and causes neurological impairments termed HIV-associated neurological disorders (HAND). HIV-associated neurotoxicity may be brought about by acute and chronic mechanisms that still remain to be fully characterized. The HIV envelope glycoprotein gp120 causes neuronal degeneration similar to that observed in HAND subjects. The present study was undertaken to discover novel mechanisms of gp120 neurotoxicity that could explain how the envelope protein promotes neurite pruning. Gp120 has been shown to associate with various intracellular organelles as well as microtubules in neurons. We then analyzed lysates of neurons exposed to gp120 with liquid chromatography mass spectrometry for potential protein interactors. We found that one of the proteins interacting with gp120 is tubulin β-3 (TUBB3), a major component of neuronal microtubules. We then tested the hypothesis that gp120 binds to neuronal microtubules. Using surface plasmon resonance we confirmed that gp120 binds with high affinity to neuronal specific TUBB3. We have also identified the binding site of gp120 to TUBB3. We then designed a small peptide (Helix-A) that displaced gp120 from binding to TUBB3. To determine whether this peptide could prevent gp120-mediated neurotoxicity, we crosslinked Helix-A to mesoporous silica nanoparticles (Helix-A nano) to enhance the intracellular delivery of the peptide. We then tested the neuroprotective property of Helix-A nano against three strains of gp120 in rat cortical neurons. Helix-A nano prevented gp120-mediated neurite simplification as well as neuronal loss. These data propose that gp120 binding to TUBB3 could be another mechanism of gp120 neurotoxicity. PMID:26826352

  19. Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation.

    PubMed

    Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A; Suh, Mi Chung; Chye, Mee-Len

    2014-10-01

    The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)-flame ionization detector (FID) and GC-mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs.

  20. Translocation and Utilization of Fungal Storage Lipid in the Arbuscular Mycorrhizal Symbiosis[w

    PubMed Central

    Bago, Berta; Zipfel, Warren; Williams, Rebecca M.; Jun, Jeongwon; Arreola, Raoul; Lammers, Peter J.; Pfeffer, Philip E.; Shachar-Hill, Yair

    2002-01-01

    The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus. PMID:11788757

  1. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

    PubMed Central

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

  2. The peroxisomal ABC transporter family.

    PubMed

    Wanders, Ronald J A; Visser, Wouter F; van Roermund, Carlo W T; Kemp, Stephan; Waterham, Hans R

    2007-02-01

    This review describes the current state of knowledge about the ABCD family of peroxisomal half adenosine-triphosphate-binding cassette (ABC) transporters. ABCDs are predicted to be present in a variety of eukaryotic organisms, although at present, only ABCDs in the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and different mammalian species have been identified and characterized to any significant extent. The functional role of none of these ABCDs has been established definitively and awaits successful reconstitution of ABCDs, either as homo- or heterodimers into liposomes, followed by transport studies. Data obtained in S. cerevisiae suggest that the two ABCDs, which have been identified in this organism, form a heterodimer, which actually transports acyl coenzyme A esters across the peroxisomal membrane. In mammals, four ABCDs have been identified, of which one [adrenoleukodystrophy protein (ALDP)] has been implicated in the transport of the coenzyme A esters of very-long-chain fatty acids. Mutations in the gene (ABCD1) encoding ALDP are the cause of a severe X-linked disease, called X-linked adrenoleukodystrophy. The availability of mutant mice in which Abcd1, Abcd2, or Abcd3 have been disrupted will help to resolve the true role of the peroxisomal half-ABC transporters.

  3. Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling 1

    PubMed Central

    Oo, Khaik Cheang; Stumpf, Paul K.

    1983-01-01

    In germinating oil palm (Elaeis guineensis var D × P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium. PMID:16663323

  4. Green tea changes serum and liver metabolomic profiles in mice with high-fat diet-induced obesity.

    PubMed

    Lee, Lan-Sook; Choi, Ji Hea; Sung, Mi Jeong; Hur, Jin-Young; Hur, Haeng Jeon; Park, Jong-Dae; Kim, Young-Chan; Gu, Eun-Ji; Min, Byungjin; Kim, Hyun-Jin

    2015-04-01

    Green tea (GT) consumption helps to prevent and control obesity by stimulating hepatic lipid metabolism. However, GT-induced changes in serum and liver metabolomes associated with the anti-obesity effects are not clearly understood. The aim of this study was to identify and validate metabolomic profiles in the livers and sera of GT-fed obese mice to elucidate the relationship between GT consumption and obesity prevention. Serum and liver metabolites were analyzed in mice fed normal diet, high-fat diet (HFD), HFD with GT, and HFD with crude catechins, using LC-quadrupole TOF MS. The addition of 1% GT to HFD reduced adipose tissue and the levels of blood triglycerides, glucose, insulin, and leptin elevated in HFD-fed mice. We proposed an HFD-induced obesity pathway and validated it by investigating the key regulatory enzymes of mitochondrial β-oxidation: carnitine palmitoyltransferase-1 and -2, acyl-coenzyme A dehydrogenase, and acetyl-coenzyme A acyltransferase. The results showed that HFD-induced abnormal mitochondrial β-oxidation was moderated by the consumption of caffeine- and theanine-enriched GT. Results of LC/MS-based metabolomic analysis of obese mice showed changes associated with abnormal lipid and energy metabolism, which were alleviated by GT intake, indicating the mechanism underlying the anti-obesity effects of GT. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Improving Fatty Acid Availability for Bio-Hydrocarbon Production in Escherichia coli by Metabolic Engineering

    PubMed Central

    Lin, Fengming; Chen, Yu; Levine, Robert; Lee, Kilho; Yuan, Yingjin; Lin, Xiaoxia Nina

    2013-01-01

    Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass (in the order of cyoA, nuoA, ndh, adhE, dld, pta, and iclR) to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation. PMID:24147139

  6. Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

    PubMed

    Padanad, Mahesh S; Konstantinidou, Georgia; Venkateswaran, Niranjan; Melegari, Margherita; Rindhe, Smita; Mitsche, Matthew; Yang, Chendong; Batten, Kimberly; Huffman, Kenneth E; Liu, Jingwen; Tang, Ximing; Rodriguez-Canales, Jaime; Kalhor, Neda; Shay, Jerry W; Minna, John D; McDonald, Jeffrey; Wistuba, Ignacio I; DeBerardinis, Ralph J; Scaglioni, Pier Paolo

    2016-08-09

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

  7. Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling1

    PubMed Central

    Pollard, Mike; Ohlrogge, John

    1999-01-01

    Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [13C218O2]Acetate was incubated with spinach (Spinacia oleracea) leaves and the 18O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an 18O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the 18O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of 18O or, less likely, complete loss of 18O, but not a 50% loss of 18O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of “prokaryotic” and “eukaryotic” lipids have both been confirmed. PMID:10594108

  8. Silencing of ACO decreases reproduction and energy metabolism in triazophos-treated female brown plant hoppers, Nilaparvata lugens Stål (Hemiptera: Delphacidae).

    PubMed

    Liu, Zong-Yu; Jiang, Yi-Ping; Li, Lei; You, Lin-Lin; Wu, You; Xu, Bin; Ge, Lin-Quan; Wu, Jin-Cai

    2016-03-01

    The brown plant hopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major pest affecting rice in Asia, and outbreaks of this pest are closely linked to pesticide-induced stimulation of reproduction. Therefore, the BPH is a classic example of a resurgent pest. However, the effects of different genes on the regulation of pesticide-induced reproductive stimulation in the BPH are unclear. In this study, the regulatory effects of acyl-coenzyme A oxidase (ACO) on the reproduction and biochemistry of the BPH were investigated with gene silencing. The number of eggs laid per female by triazophos (TZP)+dsACO BPH females was significantly lower than those of TZP-treated (without ACO silencing) or TZP+GFP females (negative control), with the number of eggs decreasing by 30.8% (from 529.5 to 366.3) and 32.0% (from 540.5 to 366.3), respectively. The preoviposition period, oviposition period, and longevity of the TZP-treated females were also influenced by dsACO treatment. Additionally, the amounts of crude fat, protein, and some fatty acids (oleic acid, palmitic acid, linoleic acid, stearic acid, and myristoleic acid) in TZP+dsACO females were significantly lower than in TZP-treated females. Thus, ACO is one of the key genes regulating the TZP-induced stimulation of reproduction in BPH females.

  9. L-carnitine for the treatment of acute myocardial infarction.

    PubMed

    Dinicolantonio, James J; Niazi, Asfandyar K; McCarty, Mark F; Lavie, Carl J; Liberopoulos, Evangelos; O'Keefe, James H

    2014-01-01

    Although the therapeutic strategies available for treating acute myocardial infarction (AMI) have evolved dramatically in recent decades, coronary artery disease remains the leading cause of death in our society, and the rates of recurrent myocardial infarction and mortality are still unacceptably high. Therefore, exploration of alternative therapeutic strategies for AMI is of utmost importance. One such strategy is to target metabolic pathways via L-carnitine supplementation. L-carnitine is a physiologically essential metabolic cofactor that has been shown to provide a plethora of benefits when administered after AMI. L-carnitine has been shown to lessen infarct size, to reduce ventricular arrhythmias, left ventricular dilation, and heart failure incidence, as well as improve survival. These benefits may, in part, be related to its ability to boost glucose oxidation in ischemic tissues, while moderating increases in fatty acyl-coenzyme A levels that can impair mitochondrial efficiency and promote oxidative stress and inflammation. This article summarizes the evidence pertinent to the therapeutic use of L-carnitine for AMI.

  10. Riboflavin-responsive lipid-storage myopathy caused by ETFDH gene mutations.

    PubMed

    Wen, Bing; Dai, Tingjun; Li, Wei; Zhao, Yuying; Liu, Shuping; Zhang, Chunhua; Li, Honghao; Wu, Jinling; Li, Danian; Yan, Chuanzhu

    2010-02-01

    Lipid-storage myopathy (LSM), defined by triglyceride accumulation in muscle fibres, is a heterogeneous group of lipid metabolic disorders predominantly affecting skeletal muscle. In the past 15 years, more than 200 cases of LSM have been reported in the Chinese literature, but the accurate pathogenic mechanisms are still unknown. In order to gain more insight into the metabolic and genetic dysfunctions of LSM, the authors described a group of Chinese patients with LSM who were very responsive to isolated riboflavin treatment (riboflavin responsive LSM, RR-LSM). Nineteen consecutive LSM patients collected during 1995-2007 in our Neuromuscular Laboratory who were dramatically responsive to riboflavin and presented with proximal muscle weakness, exercise intolerance and elevated serum CK but without episodic encephalopathy were subjected to pathological, biochemical and molecular analysis. On the basis of muscle pathology, all 19 patients were diagnosed as LSM. Seventeen patients were suspected of having multiple acyl-coenzyme A dehydrogenase deficiency (MADD) according to blood acylcarnitine profiles and urine organic acid analysis. Genetic analysis identified 19 novel mutations in ETFDH gene in 18 patients, among which one was homozygote, 16 were compound heterozygotes, and one was a single heterozygote. No pathogenic mutation was detected in ETFA or ETFB genes. Western blot analysis showed there was no significant decrease in ETF:QO expression except for one patient. The research findings suggest that the majority of Chinese patients with RR-LSM are caused by a mild type of MADD with unique myopathy which is due to ETFDH gene mutation.

  11. Experimental evidence for protein oxidative damage and altered antioxidant defense in patients with medium-chain acyl-CoA dehydrogenase deficiency.

    PubMed

    Derks, Terry G J; Touw, Catharina M L; Ribas, Graziela S; Biancini, Giovana B; Vanzin, Camila S; Negretto, Giovanna; Mescka, Caroline P; Reijngoud, Dirk Jan; Smit, G Peter A; Wajner, Moacir; Vargas, Carmen R

    2014-09-01

    The objective of this study was to test whether macromolecule oxidative damage and altered enzymatic antioxidative defenses occur in patients with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. We performed a cross-sectional observational study of in vivo parameters of lipid and protein oxidative damage and antioxidant defenses in asymptomatic, nonstressed, MCAD-deficient patients and healthy controls. Patients were subdivided into three groups based on therapy: patients without prescribed supplementation, patients with carnitine supplementation, and patients with carnitine plus riboflavin supplementation. Compared with healthy controls, nonsupplemented MCAD-deficient patients and patients receiving carnitine supplementation displayed decreased plasma sulfhydryl content (indicating protein oxidative damage). Increased erythrocyte superoxide dismutase (SOD) activity in patients receiving carnitine supplementation probably reflects a compensatory mechanism for scavenging reactive species formation. The combination of carnitine plus riboflavin was not associated with oxidative damage. These are the first indications that MCAD-deficient patients experience protein oxidative damage and that combined supplementation of carnitine and riboflavin may prevent these biochemical alterations. Results suggest involvement of free radicals in the pathophysiology of MCAD deficiency. The underlying mechanisms behind the increased SOD activity upon carnitine supplementation need to be determined. Further studies are necessary to determine the clinical relevance of oxidative stress, including the possibility of antioxidant therapy.

  12. Brown-Vialetto-Van Laere syndrome: a riboflavin-unresponsive patient with a novel mutation in the C20orf54 gene.

    PubMed

    Koy, Anne; Pillekamp, Frank; Hoehn, Thomas; Waterham, Hans; Klee, Dirk; Mayatepek, Ertan; Assmann, Birgit

    2012-06-01

    Brown-Vialetto-Van Laere syndrome (Online Mendelian Inheritance in Man number 211530) is a neurodegenerative disorder characterized by pontobulbar palsy affecting cranial nerves (mainly VII-XII). Sensorineural deafness is often the leading sign, followed by other neurologic signs. Inheritance is often autosomal recessive, with mutations in the C20orf54 gene (Online Mendelian Inheritance in Man number 613350). Three previous patients with mutations in the C20orf54 gene and clinical signs of Brown-Vialetto-Van Laere or Fazio-Londe syndrome revealed a metabolic profile suggesting a multiple acyl-coenzyme A dehydrogenase defect. They benefited from riboflavin. We describe a 3-year-old girl with early-onset Brown-Vialetto-Van Laere syndrome and a novel mutation in the C20orf54 gene (c.989G>T). On T(2)-weighted imaging, increased signal intensity of the vestibular nuclei bilaterally, the pedunculus cerebellaris superior and the central tegmental tract were observed during acute clinical deterioration. Her metabolic profile was normal. Trials with steroids, immunoglobulins, and riboflavin produced no effect. The patient recovered slowly during subsequent months, with residual deficits. Brown-Vialetto-Van Laere syndrome should be considered in patients with sensorineural hearing loss and pontobulbar palsy. Patients should be screened for riboflavin deficiency and a therapy with riboflavin may provide effective treatment in some affected patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean.

    PubMed

    Harryson, P.; Morre, D. J.; Sandelius, A. S.

    1996-02-01

    Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.

  14. Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean.

    PubMed Central

    Harryson, P.; Morre, D. J.; Sandelius, A. S.

    1996-01-01

    Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity. PMID:12226209

  15. Differential effects of triclosan on the activation of mouse and human peroxisome proliferator-activated receptor alpha.

    PubMed

    Wu, Yuanfeng; Wu, Qiangen; Beland, Frederick A; Ge, Peter; Manjanatha, Mugimane G; Fang, Jia-Long

    2014-11-18

    Triclosan is an anti-bacterial agent used in many personal care products, household items, medical devices, and clinical settings. Liver tumors occur in mice exposed to triclosan, a response attributed to peroxisome proliferator-activated receptor alpha (PPARα) activation; however, the effects of triclosan on mouse and human PPARα have not been fully evaluated. We compared the effects of triclosan on mouse and human PPARα using PPARα reporter assays and on downstream events of PPARα activation using mouse hepatoma Hepa1c1c7 cells and human hepatoma HepG2 cells. PPARα transcriptional activity was increased by triclosan in a mouse PPARα reporter assay and decreased in a human PPARα reporter assay. Concentrations of triclosan inhibiting 50% cell growth were similar in both human and mouse hepatoma cells. Western blotting analysis showed that triclosan increased acyl-coenzyme A oxidase (ACOX1), a PPARα target, in Hepa1c1c7 cells but decreased the level in HepG2 cells. Treatment of Hepa1c1c7 cells with triclosan enhanced DNA synthesis and suppressed transforming growth factor beta-mediated apoptosis. This did not occur in HepG2 cells. These data demonstrate that triclosan had similar cytotoxicity in Hepa1c1c7 and HepG2 cells, but differential effects on the activation of PPARα, the expression of ACOX1, and downstream events including DNA synthesis and apoptosis.

  16. Research for Developing Renewable Biofuels from Algae

    SciTech Connect

    Black, Paul N.

    2012-12-15

    Task A. Expansion of knowledge related to lipid production and secretion in algae A.1 Lipid biosynthesis in target algal species; Systems biology approaches are being used in combination with recent advances in Chlorella and Chlamydomonas genomics to address lipid accumulation in response to defined nutrient regimes. The UNL Algal Group continues screening additional species of Chlorella and other naturally occurring algae for those with optimal triglyceride production; Of the strains examined by the DOE's Aquatic Species Program, green algae, several species of Chlorella represent the largest group from which oleaginous candidates have been identified; A.1.1. Lipid profiling; Neutral lipid accumulation is routinely monitored by Nile red and BODIPY staining using high throughput strategies to screen for naturally occurring algae that accumulate triglyceride. These strategies complement those using spectrofluorometry to quantify lipid accumulation; Neutral lipid accumulation is routinely monitored by high performance thin-layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) of lipid extracts in conjunction with; Carbon portioning experiments have been completed and the data currently are being analyzed and prepared for publication; Methods in the Black lab were developed to identify and quantify triacylglycerol (TAG), major membrane lipids [diacylglycerol trimethylhomoserine, phosphatidylethanolamine and chloroplast glycolipids], biosynthetic intermediates such as diacylglycerol, phosphatidic acid and lysophospholipids and different species of acyl-coenzyme A (acyl CoA).

  17. Macrophage-mediated cholesterol handling in atherosclerosis.

    PubMed

    Chistiakov, Dimitry A; Bobryshev, Yuri V; Orekhov, Alexander N

    2016-01-01

    Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

  18. Acylcarnitines--old actors auditioning for new roles in metabolic physiology.

    PubMed

    McCoin, Colin S; Knotts, Trina A; Adams, Sean H

    2015-10-01

    Perturbations in metabolic pathways can cause substantial increases in plasma and tissue concentrations of long-chain acylcarnitines (LCACs). For example, the levels of LCACs and other acylcarnitines rise in the blood and muscle during exercise, as changes in tissue pools of acyl-coenzyme A reflect accelerated fuel flux that is incompletely coupled to mitochondrial energy demand and capacity of the tricarboxylic acid cycle. This natural ebb and flow of acylcarnitine generation and accumulation contrasts with that of inherited fatty acid oxidation disorders (FAODs), cardiac ischaemia or type 2 diabetes mellitus. These conditions are characterized by very high (FAODs, ischaemia) or modestly increased (type 2 diabetes mellitus) tissue and blood levels of LCACs. Although specific plasma concentrations of LCACs and chain-lengths are widely used as diagnostic markers of FAODs, research into the potential effects of excessive LCAC accumulation or the roles of acylcarnitines as physiological modulators of cell metabolism is lacking. Nevertheless, a growing body of evidence has highlighted possible effects of LCACs on disparate aspects of pathophysiology, such as cardiac ischaemia outcomes, insulin sensitivity and inflammation. This Review, therefore, aims to provide a theoretical framework for the potential consequences of tissue build-up of LCACs among individuals with metabolic disorders.

  19. A diagnostic algorithm for metabolic myopathies.

    PubMed

    Berardo, Andres; DiMauro, Salvatore; Hirano, Michio

    2010-03-01

    Metabolic myopathies comprise a clinically and etiologically diverse group of disorders caused by defects in cellular energy metabolism, including the breakdown of carbohydrates and fatty acids to generate adenosine triphosphate, predominantly through mitochondrial oxidative phosphorylation. Accordingly, the three main categories of metabolic myopathies are glycogen storage diseases, fatty acid oxidation defects, and mitochondrial disorders due to respiratory chain impairment. The wide clinical spectrum of metabolic myopathies ranges from severe infantile-onset multisystemic diseases to adult-onset isolated myopathies with exertional cramps. Diagnosing these diverse disorders often is challenging because clinical features such as recurrent myoglobinuria and exercise intolerance are common to all three types of metabolic myopathy. Nevertheless, distinct clinical manifestations are important to recognize as they can guide diagnostic testing and lead to the correct diagnosis. This article briefly reviews general clinical aspects of metabolic myopathies and highlights approaches to diagnosing the relatively more frequent subtypes (Fig. 1). Fig. 1 Clinical algorithm for patients with exercise intolerance in whom a metabolic myopathy is suspected. CK-creatine kinase; COX-cytochrome c oxidase; CPT-carnitine palmitoyl transferase; cyt b-cytochrome b; mtDNA-mitochondrial DNA; nDNA-nuclear DNA; PFK-phosphofructokinase; PGAM-phosphoglycerate mutase; PGK-phosphoglycerate kinase; PPL-myophosphorylase; RRF-ragged red fibers; TFP-trifunctional protein deficiency; VLCAD-very long-chain acyl-coenzyme A dehydrogenase.

  20. Plasma Metabolomics Biosignature According to HIV Stage of Infection, Pace of Disease Progression, Viremia Level and Immunological Response to Treatment

    PubMed Central

    Scarpelini, Bruno; Zanoni, Michelle; Sucupira, Maria Cecilia Araripe; Truong, Hong-Ha M.; Janini, Luiz Mario Ramos; Segurado, Ismael Dale Cotrin; Diaz, Ricardo Sobhie

    2016-01-01

    Background We evaluated plasma samples HIV-infected individuals with different phenotypic profile among five HIV-infected elite controllers and five rapid progressors after recent HIV infection and one year later and from 10 individuals subjected to antiretroviral therapy, five of whom were immunological non-responders (INR), before and after one year of antiretroviral treatment compared to 175 samples from HIV-negative patients. A targeted quantitative tandem mass spectrometry metabolomics approach was used in order to determine plasma metabolomics biosignature that may relate to HIV infection, pace of HIV disease progression, and immunological response to treatment. Results Twenty-five unique metabolites were identified, including five metabolites that could distinguish rapid progressors and INRs at baseline. Severe deregulation in acylcarnitine and sphingomyelin metabolism compatible with mitochondrial deficiencies was observed. β-oxidation and sphingosine‐1‐phosphate-phosphatase-1 activity were down-regulated, whereas acyl-alkyl-containing phosphatidylcholines and alkylglyceronephosphate synthase levels were elevated in INRs. Evidence that elite controllers harbor an inborn error of metabolism (late-onset multiple acyl-coenzyme A dehydrogenase deficiency [MADD]) was detected. Conclusions Blood-based markers from metabolomics show a very high accuracy of discriminating HIV infection between varieties of controls and have the ability to predict rapid disease progression or poor antiretroviral immunological response. These metabolites can be used as biomarkers of HIV natural evolution or treatment response and provide insight into the mechanisms of the disease. PMID:27941971

  1. Firefly bioluminescence: a mechanistic approach of luciferase catalyzed reactions.

    PubMed

    Marques, Simone M; Esteves da Silva, Joaquim C G

    2009-01-01

    Luciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH(2)), with ATP-Mg(2+) generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH(2)-AMP); the second is the oxidation and decarboxylation of LH(2)-AMP to oxyluciferin, the light emitter, producing CO(2), AMP, and photons of yellow-green light (550- 570 nm). In a dark reaction LH(2)-AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH(2)-CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3'-phosphate chain plus an intact terminal pyrophosphate moiety. The LH(2) stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH(2) or L-LH(2), has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.

  2. Effects of a gut pathobiont in a gnotobiotic mouse model of childhood undernutrition.

    PubMed

    Wagner, Vitas E; Dey, Neelendu; Guruge, Janaki; Hsiao, Ansel; Ahern, Philip P; Semenkovich, Nicholas P; Blanton, Laura V; Cheng, Jiye; Griffin, Nicholas; Stappenbeck, Thaddeus S; Ilkayeva, Olga; Newgard, Christopher B; Petri, William; Haque, Rashidul; Ahmed, Tahmeed; Gordon, Jeffrey I

    2016-11-23

    To model how interactions among enteropathogens and gut microbial community members contribute to undernutrition, we colonized gnotobiotic mice fed representative Bangladeshi diets with sequenced bacterial strains cultured from the fecal microbiota of two 24-month-old Bangladeshi children: one healthy and the other underweight. The undernourished donor's bacterial collection contained an enterotoxigenic Bacteroides fragilis strain (ETBF), whereas the healthy donor's bacterial collection contained two nontoxigenic strains of B. fragilis (NTBF). Analyses of mice harboring either the unmanipulated culture collections or systematically manipulated versions revealed that ETBF was causally related to weight loss in the context of its native community but not when introduced into the healthy donor's community. This phenotype was transmissible from the dams to their offspring and was associated with derangements in host energy metabolism manifested by impaired tricarboxylic acid cycle activity and decreased acyl-coenzyme A utilization. NTBF reduced ETBF's expression of its enterotoxin and mitigated the effects of ETBF on the transcriptomes of other healthy donor community members. These results illustrate how intraspecific (ETBF-NTBF) and interspecific interactions influence the effects of harboring B. fragilis.

  3. Short-term lab exposures of immature rainbow trout (Oncorhynchus mykiss) to sulfite and kraft pulp-mill effluents: effects on oxidative stress and circulating sex steroids.

    PubMed

    Oakes, Ken D; Tremblay, Louis A; van der Kraak, Glen J

    2005-06-01

    This study investigates the temporal onset of reactive oxygen species (ROS) damage and changes in circulating sex steroids in immature rainbow trout exposed over 21 d to two pulp-mill effluents. Exposure to effluent from a bleached sulfite mill produced increases in 2-thiobarbituric acid reactive substances (TBARS), ethoxyresorufin O-deethylase (EROD) activity, hepatic free iron, and significant depressions in hepatic ascorbic acid. Impairments in pregnenolone production relative to cholesterol availability suggest an effect of sulfite-mill effluent early in the steroidogenic pathway. Induction of vitellogenin in immature fish exposed to effluent from this mill, relative to waterborne 17 beta-estradiol treatments, indicated sulfite-mill effluent contained constituents capable of binding the estrogen receptor. Exposure to a kraft-mill effluent also elevated hepatic TBARS, tissue normalized fatty acyl-coenzyme A oxidase (FAO) activity, and hepatic free iron while producing commensurate declines in hepatic ascorbic acid. Plasma testosterone, 11-ketotestosterone, and 17 beta-estradiol were elevated with kraft-mill effluent exposure, but no changes in vitellogenin induction were observed. In summary, effluent from bleached sulfite and bleached kraft mills yielded similar oxidative stress responses, but marked differences were observed in the endocrine-disrupting potential of each effluent.

  4. Novel N-terminal cleavage of APP precludes Abeta generation in ACAT-defective AC29 cells.

    PubMed

    Huttunen, Henri J; Puglielli, Luigi; Ellis, Blake C; MacKenzie Ingano, Laura A; Kovacs, Dora M

    2009-01-01

    A common pathogenic event that occurs in all forms of Alzheimer's disease is the progressive accumulation of amyloid beta-peptide (Abeta) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Abeta from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both alpha and the amyloidogenic beta cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Abeta generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Abeta generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment.

  5. Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

    PubMed Central

    Müller, Jana; MacEachran, Daniel; Burd, Helcio; Sathitsuksanoh, Noppadon; Bi, Changhao; Yeh, Yi-Chun; Lee, Taek Soon; Hillson, Nathan J.; Chhabra, Swapnil R.; Singer, Steven W.

    2013-01-01

    Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones. PMID:23686271

  6. Dictyostelium discoideum Dgat2 can substitute for the essential function of Dgat1 in triglyceride production but not in ether lipid synthesis.

    PubMed

    Du, Xiaoli; Herrfurth, Cornelia; Gottlieb, Thomas; Kawelke, Steffen; Feussner, Kristin; Rühling, Harald; Feussner, Ivo; Maniak, Markus

    2014-04-01

    Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

  7. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    PubMed

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  8. Synthesis of medium chain length fatty acid ethyl esters in engineered Escherichia coli using endogenously produced medium chain fatty acids.

    PubMed

    Fan, Liping; Liu, Junfeng; Nie, Kaili; Liu, Luo; Wang, Fang; Tan, Tianwei; Deng, Li

    2013-07-10

    Microbial biosynthesis of fatty acid-derived biofuels from renewable carbon sources has attracted significant attention in recent years. Free fatty acids (FFAs) can be used as precursors for the production of micro-diesel. The expression of codon optimized two plants (Umbellularia californica and Cinnamomum camphora) medium-chain acyl-acyl carrier protein (ACP) thioesterase genes (ucFatB and ccFatB) in Escherichia coli resulted in a very high level of extractable medium-chain-specific hydrolytic activity and caused large accumulation of medium-chain free fatty acids. By heterologous co-expression of acyl-coenzyme A:diacylglycerol acyltransferase from Acinetobacter baylyi ADP1, specific plant thioesterases in E. coli, with supplementation of exogenous ethanol, resulted in drastic changes in fatty acid ethyl esters (FAEEs) composition ranging from 12:0 to 18:1. Through an optimized microbial shake-flask fermentation of two modified E. coli strains, yielded FFAs and FAEEs in the concentration of approximately 500 mg L(-1)/250 mg L(-1) and 2.01 mg g(-1)/1.99 mg g(-1), respectively. The optimal ethanol level for FAEEs yield in the two recombinant strains was reached at the 3% ethanol concentration, which was about 5.4-fold and 1.93-fold higher than that of 1% ethanol concentration. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

    PubMed Central

    Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-01-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

  10. Transport and function of L-carnitine and L-propionylcarnitine: relevance to some cardiomyopathies and cardiac ischemia.

    PubMed

    Siliprandi, N; Di Lisa, F; Pivetta, A; Miotto, G; Siliprandi, D

    1987-01-01

    Carnitine, an essential cofactor in fatty acid oxidation, plays a central role in myocardial metabolism. Interpretation of the biochemical features of disturbed myocardial function, particularly in ischemia, may be facilitated by understanding carnitine biosynthesis, transport and function. Biosynthesis: In man, deoxycarnitine, the immediate precursor of carnitine, is synthesized in all tissues, whereas the last step, the conversion of deoxycarnitine into carnitine may only take place in liver, kidney and brain (Figs. 1 and 2). Deoxycarnitine formed by organs like muscle or heart is released into the plasma, taken up by liver and kidney, converted into carnitine which is secreted into the bloodstream to be taken up by heart or muscle (Fig. 2). Carnitine transport and cellular function: The myocardial uptake of carnitine against a large concentration gradient (Table 1) occurs in an 1:1 exchange-diffusion process. Under physiological conditions, intracellular deoxycarnitine is exported and extracellular carnitine is imported. According to this model, myocardial carnitine deficiency may be due either to a functional alteration of the sarcolemmal carnitine carrier or to a deficient synthesis of deoxycarnitine. D-carnitine, acetylcarnitine and long-chain acylcarnitine esters are also transported by the carrier at different rates. This might account for the release of endogenous acylcarnitines accumulated in anoxic or ischemic conditions, contributing to the cardioprotective effect of carnitine by reduction in intracellular long-chain acyl-coenzyme A.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

    PubMed

    Waché, Y; Aguedo, M; Choquet, A; Gatfield, I L; Nicaud, J M; Belin, J M

    2001-12-01

    Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.

  12. Dissection of the phytohormonal regulation of trichome formation and biosynthesis of the antimalarial compound artemisinin in Artemisia annua plants.

    PubMed

    Maes, Lies; Van Nieuwerburgh, Filip C W; Zhang, Yansheng; Reed, Darwin W; Pollier, Jacob; Vande Casteele, Sofie R F; Inzé, Dirk; Covello, Patrick S; Deforce, Dieter L D; Goossens, Alain

    2011-01-01

    • Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

  13. De novo Biosynthesis of Biodiesel by Escherichia coli in Optimized Fed-Batch Cultivation

    PubMed Central

    Cai, Ke; Tan, Xiaoming; Lu, Xuefeng

    2011-01-01

    Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate. PMID:21629774

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

    PubMed

    Cao, Yunpeng; Han, Yahui; Li, Dahui; Lin, Yi; Cai, Yongping

    2016-10-19

    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.

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

    PubMed Central

    Cao, Yunpeng; Han, Yahui; Li, Dahui; Lin, Yi; Cai, Yongping

    2016-01-01

    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. PMID:27775579

  16. Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio.

    PubMed

    Takahashi, Toshinari; Ohara, Yusuke; Sueno, Kazuo

    2017-03-09

    Sake yeast produces a fruity flavor known as ginjo-ko-which is mainly attributable to ethyl caproate and isoamyl acetate-during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing us