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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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Acyl-coenzyme A binding domain containing 3 (ACBD3; PAP7; GCP60): an emerging signaling molecule

    PubMed Central

    Fan, Jinjiang; Liu, Jun; Culty, Martine; Papadopoulos, Vassilios

    2010-01-01

    Golgi body-mediated signaling has been linked to its fragmentation and regeneration during the mitotic cycle of the cell. During this process, Golgi-resident proteins are released to the cytosol and interact with other signaling molecules to regulate various cellular processes. Acyl-coenzyme A binding domain containing 3 protein (ACBD3) is a Golgi protein involved in several signaling events. ACBD3 protein was previously known as peripheral-type benzodiazepine receptor and cAMP-dependent protein kinase associated protein 7 (PAP7), Golgi complex-associated protein of 60 kDa (GCP60), Golgi complex-associated protein 1 (GOCAP1), and Golgi phosphoprotein 1 (GOLPH1). In this review, we present the gene ontology of ACBD3, its relations to other Acyl-coenzyme A binding protein (ACBP) domain containing proteins, and its biological function in steroidogenesis, apoptosis, neurogenesis, and embryogenesis. We also discuss the role of ACBD3 in asymmetric cell division and cancer. New findings about ACBD3 may help understand this newly characterized signaling molecule and stimulate further research into its role in molecular endocrinology, neurology, and stem cell biology. PMID:20043945

  3. Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

    PubMed Central

    Zhang, Jiantao; Diaz, Arturo; Mao, Lan; Ahlquist, Paul

    2012-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly. PMID:22345450

  4. Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p

    PubMed Central

    Rijken, Pieter J.; Houtkooper, Riekelt H.; Akbari, Hana; Brouwers, Jos F.; Koorengevel, Martijn C.; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M.; de Kroon, Anton I. P. M.

    2009-01-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  5. 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

  6. Cardiolipin molecular species with shorter acyl chains accumulate in Saccharomyces cerevisiae mutants lacking the acyl coenzyme A-binding protein Acb1p: new insights into acyl chain remodeling of cardiolipin.

    PubMed

    Rijken, Pieter J; Houtkooper, Riekelt H; Akbari, Hana; Brouwers, Jos F; Koorengevel, Martijn C; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M; de Kroon, Anton I P M

    2009-10-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  7. Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans▿

    PubMed Central

    Meng, Yu; Ingram-Smith, Cheryl; Cooper, Leroy L.; Smith, Kerry S.

    2010-01-01

    Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated MacsMa, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PPi were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PPi. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The MacsMa structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys256, Cys298, Gly351, Trp259, Trp237, and Trp254, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme. PMID:20851904

  8. Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase.

    PubMed Central

    Buszczak, Michael; Lu, Xiaohui; Segraves, William A; Chang, Ta Yuan; Cooley, Lynn

    2002-01-01

    During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila. PMID:11973306

  9. Prolonged QTc interval in association with medium-chain acyl-coenzyme A dehydrogenase deficiency.

    PubMed

    Wiles, Jason R; Leslie, Nancy; Knilans, Timothy K; Akinbi, Henry

    2014-06-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxidation. We report a term male infant who presented at 3 days of age with hypoglycemia, compensated metabolic acidosis, hypocalcemia, and prolonged QTc interval. Pregnancy was complicated by maternal premature atrial contractions and premature ventricular contractions. Prolongation of the QTc interval resolved after correction of metabolic derangements. The newborn screen was suggestive for MCAD deficiency, a diagnosis that was confirmed on genetic analysis that showed homozygosity for the disease-associated missense A985G mutation in the ACADM gene. This is the first report of acquired prolonged QTc in a neonate with MCAD deficiency, and it suggests that MCAD deficiency should be considered in the differential diagnoses of acute neonatal illnesses associated with electrocardiographic abnormality. We review the clinical presentation and diagnosis of MCAD deficiency in neonates. PMID:24799540

  10. 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

  11. Acyl-coenzyme A:cholesterol O-acyltransferase is not identical to liver microsomal carboxylesterase.

    PubMed

    Diczfalusy, M A; Björkhem, I; Einarsson, K; Alexson, S E

    1996-04-01

    Acyl-coenzyme A (CoA):cholesterol O-acyltransferase (ACAT) is responsible for esterification of cholesterol in the cell. The enzyme has never been purified, but two cDNA sequences coding for this enzyme were recently reported. One of the sequences was identical to human liver carboxylesterase. We have used inhibitors to elucidate the relation between microsomal carboxylesterase, acyl-CoA hydrolase (ACH), and ACAT activities in rat liver. Low concentrations of serine esterase inhibitors strongly inhibited carboxylesterase and acyl-CoA hydrolase activities but stimulated ACAT activity. At higher concentrations, ACAT activity was also inhibited. A sulfhydryl-modifying agent was found to be a potent inhibitor of ACAT without affecting carboxylesterase activity. Similarly, two specific ACAT inhibitors, DL-melinamide and PD 138142-15, inhibited ACAT activity but did not affect carboxylesterase or ACH activities. Our data thus exclude ACAT as a liver microsomal carboxylesterase. The complex inhibition patterns observed with serine esterase inhibitors indicate that carboxylesterases and ACHs may interfere with ACAT activity by competing for the substrate. It is obvious that final identification of ACAT requires demonstration of an active homogenous protein. PMID:8624784

  12. Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes.

    PubMed

    Bavdek, Andrej; Vazquez, Hector M; Conzelmann, Andreas

    2015-11-01

    Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [(3)H]lyso-phosphatidic acid inside liposomes after [(3)H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [(3)H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [(3)H]lyso-phosphatidic acid or of dephosphorylated [(3)H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required. PMID:26325346

  13. 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

  14. 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

  15. 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. PMID:16638618

  16. Identification of a Long-Chain Polyunsaturated Fatty Acid Acyl-Coenzyme A Synthetase from the Diatom Thalassiosira pseudonana1

    PubMed Central

    Tonon, Thierry; Qing, Renwei; Harvey, David; Li, Yi; Larson, Tony Robert; Graham, Ian Alexander

    2005-01-01

    The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast. PMID:15821149

  17. Purification of Recombinant Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (ACAT1) from H293 Cells and Binding Studies Between the Enzyme and Substrates Using Difference Intrinsic Fluorescence Spectroscopy†

    PubMed Central

    Chang, Catherine CY; Miyazaki, Akira; Dong, Ruhong; Kheirollah, Alireza; Yu, Chunjiang; Geng, Yong; Higgs, Henry N; Chang, Ta-Yuan

    2010-01-01

    Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl CoA binds to ACAT1 with Kd=1.9 μM, and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl CoA elicits a similar spectrum change with much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3beta-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1. PMID:20964445

  18. Fatty Acid Export from the Chloroplast. Molecular Characterization of a Major Plastidial Acyl-Coenzyme A Synthetase from Arabidopsis1

    PubMed Central

    Schnurr, Judy A.; Shockey, Jay M.; de Boer, Gert-Jan; Browse, John A.

    2002-01-01

    Acyl-coenzyme A (CoA) synthetases (ACSs, EC 6.2.1.3) catalyze the formation of fatty acyl-CoAs from free fatty acid, ATP, and CoA. Essentially all de novo fatty acid synthesis occurs in the plastid. Fatty acids destined for membrane glycerolipid and triacylglycerol synthesis in the endoplasmic reticulum must be first activated to acyl-CoAs via an ACS. Within a family of nine ACS genes from Arabidopsis, we identified a chloroplast isoform, LACS9. LACS9 is highly expressed in developing seeds and young rosette leaves. Both in vitro chloroplast import assays and transient expression of a green fluorescent protein fusion indicated that the LACS9 protein is localized in the plastid envelope. A T-DNA knockout mutant (lacs9-1) was identified by reverse genetics and these mutant plants were indistinguishable from wild type in growth and appearance. Analysis of leaf lipids provided no evidence for compromised export of acyl groups from chloroplasts. However, direct assays demonstrated that lacs9-1 plants contained only 10% of the chloroplast long-chain ACS activity found for wild type. The residual long-chain ACS activity in mutant chloroplasts was comparable with calculated rates of fatty acid synthesis. Although another isozyme contributes to the activation of fatty acids during their export from the chloroplast, LACS9 is a major chloroplast ACS. PMID:12177483

  19. 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

  20. Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus1

    PubMed Central

    Hlousek-Radojcic, Alenka; Evenson, Kimberly J.; Jaworski, Jan G.; Post-Beittenmiller, Dusty

    1998-01-01

    In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

  1. 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.

  2. Cloning and functional analysis of human acyl coenzyme A: Cholesterol acyltransferase1 gene P1 promoter.

    PubMed

    Ge, Jing; Cheng, Bei; Qi, Benling; Peng, Wen; Wen, Hui; Bai, Lijuan; Liu, Yun; Zhai, Wei

    2016-07-01

    Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) catalyzes the conversion of free cholesterol (FC) to cholesterol ester. The human ACAT1 gene P1 promoter has been cloned. However, the activity and specificity of the ACAT1 gene P1 promoter in diverse cell types remains unclear. The P1 promoter fragment was digested with KpnI/XhoI from a P1 promoter cloning vector, and was subcloned into the multiple cloning site of the Firefly luciferase vector pGL3‑Enhancer to obtain the construct P1E‑1. According to the analysis of biological information, the P1E‑1 plasmid was used to generate deletions of the ACAT1 gene P1 promoter with varying 5' ends and an identical 3' end at +65 by polymerase chain reaction (PCR). All the 5'‑deletion constructs of the P1 promoter were identified by PCR, restriction enzyme digestion mapping and DNA sequencing. The transcriptional activity of each construct was detected after transient transfection into THP‑1, HepG2, HEK293 and Hela cells using DEAE‑dextran and Lipofectamine 2000 liposome transfection reagent. Results showed that the transcriptional activity of the ACAT1 gene P1 promoter and deletions of P1 promoter in THP‑1 and HepG2 cells was higher than that in HEK293 and HeLa cells. Moreover, the transcriptional activity of P1E‑9 was higher compared with those of other deletions in THP‑1, HepG2, HEK293 and HeLa cells. These findings indicate that the transcriptional activity of the P1 promoter and the effects of deletions vary with different cell lines. Thus, the P1 promoter may drive ACAT1 gene expression with cell‑type specificity. In addition, the core sequence of ACAT1 gene P1 promoter was suggested to be between -125 and +65 bp. PMID:27220725

  3. Levels of acyl-coenzyme A synthetase 5 in urothelial cells and corresponding neoplasias reflect cellular differentiation.

    PubMed

    Gaisa, Nadine T; Reinartz, Andrea; Schneider, Ursula; Klaus, Christina; Heidenreich, Axel; Jakse, Gerhard; Kaemmerer, Elke; Klinkhammer, Barbara Mara; Knuechel, Ruth; Gassler, Nikolaus

    2013-03-01

    Metabolic components like fatty acids and acyl-Coenzyme A (acyl-CoA) thioesters have been implicated in the pathogenesis of various tumours. The activation of fatty acids to acyl-CoAs is catalysed by long chain acyl-CoA synthetases (ACSLs), and impairment of ACSL expression levels has been associated with tumourigenesis and progression. Since ACSLs have never been investigated in bladder tissues, the study aims to characterize ACSL expression and acyl-CoA synthesis in normal and neoplastic bladder tissues, as well as cell lines. ACSL isoforms 1, 3, 4 and 5 and synthesis of acyl-CoAs were analysed using qRT-PCR, western blot analysis, immunohistochemistry and lipid mass spectrometry. In normal urothelium, expression of ACSL1, 3, 4 and 5, with highest levels of ACSL isoform 5 was found. However, ACSL5 expression was reduced in corresponding neoplastic tissues and urothelial cell lines depending on the grade of cellular differentiation. Anti-ACSL5 immunostainings showed expression in normal urothelium and a gradual loss of ACSL5 protein via pre-invasive lesions to invasive carcinomas. High expression of ACSL5 correlated with increased α-galactosidase activity and positive Uroplakin III staining in tumours. In contrast, synthesis of acyl-CoAs was enhanced in neoplastic bladder tissues compared to normal urothelium, and reflected an increase with respect to cellular differentiation. These results confirm an expression of ACSLs, especially isoform 5, in human urothelium, prove enzymatic/lipidomic changes in bladder cancer tissues, and suggest an involvement of ACSL5 in cellular maturation and/or senescence with possible effects onto induction of tumour formation or progression. Further work may identify responsible pathway alterations, and attempting to re-balance the metabolic equilibrium of the urothelium may offer a further opportunity for tumour treatment and prevention. PMID:23348389

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. The Acyl-Coenzyme A:Cholesterol Acyltransferase Inhibitor CI-1011 Reverses Diffuse Brain Amyloid Pathology in Aged Amyloid Precursor Protein Transgenic Mice

    PubMed Central

    Huttunen, Henri J.; Havas, Daniel; Peach, Camilla; Barren, Cory; Duller, Stephan; Xia, Weiming; Frosch, Matthew P.; Hutter-Paier, Birgit; Windisch, Manfred; Kovacs, Dora M.

    2010-01-01

    Cerebral accumulation of amyloid β-peptide (Aβ) is characteristic of Alzheimer disease and of amyloid precursor protein (APP) transgenic mice. Here, we assessed the efficacy of CI-1011, an inhibitor of acyl-coenzyme A:cholesterol acyltransferase, which is suitable for clinical use, in reducing amyloid pathology in both young (6.5 months old) and aged (16 months old) hAPP transgenic mice. Treatment of young animals with CI-1011 decreased amyloid plaque load in the cortex and hippocampus and reduced the levels of insoluble Aβ40 and Aβ42 and C-terminal fragments of APP in brain extracts. In aged mice, CI-1011 specifically reduced diffuse amyloid plaques with a minor effect on thioflavin S+ dense-core plaques. Reduced diffusible amyloid was accompanied by suppression of astrogliosis and enhanced microglial activation. Collectively, these data suggest that CI-1011 treatment reduces amyloid burden in hAPP mice by limiting generation and increasing clearance of diffusible Aβ. PMID:20613640

  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. PMID:15678619

  11. 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. PMID:25468933

  12. 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

  13. Modulation Peroxisome Proliferators Activated Receptor alpha (PPAR α) and Acyl Coenzyme A: Cholesterol Acyltransferase1 (ACAT1) Gene expression by Fatty Acids in Foam cell

    PubMed Central

    Zavvar Reza, Javad; Doosti, Mahmoud; salehipour, Masoud; PackneJad, Malehieh; Mojarrad, Majed; Heidari, Mansour; Emamian, Effat S

    2009-01-01

    Background One of the most important factors in the initiation and progression of atherosclerosis is the default in macrophage cholesterol homeostasis. Many genes and transcription factors such as Peroxisome Proliferators Activated Receptors (PPARs) and Acyl Coenzyme A: Cholesterol Acyltransferase1 (ACAT1) are involved in cholesterol homeostasis. Fatty Acids are important ligands of PPARα and the concentration of them can effect expression of ACAT1. So this study designed to clarified on the role of these genes and fatty acids on the lipid metabolism in foam cells. Methods This study examined effects of c9, t11-Conjugated Linoleic Acid(c9, t11-CLA), Alpha Linolenic Acid (LA), Eicosapentaenoic Acid (EPA) on the PPARα and ACAT1 genes expression by using Real time PCR and cholesterol homeostasis in THP-1 macrophages derived foam cells. Results Incubation of c9, t11-CLA, LA cause a significant reduction in intracellular Total Cholesterol, Free Cholesterol, cellular and Estrified Cholesterol concentrations (P ≤ 0.05). CLA and LA had no significant effect on the mRNA levels of ACAT1, but EPA increased ACAT1 mRNA expression (P = 0.003). Treatment with EPA increased PPARα mRNA levels (P ≤ 0.001), although CLA, LA had no significant effect on PPARα mRNA expression. Conclusion In conclusion, it seems that different fatty acids have different effects on gene expression and lipid metabolism and for complete conception study of the genes involved in lipid metabolism in foam cell all at once maybe is benefit. PMID:19725980

  14. The Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase from Acinetobacter sp. Strain ADP1: Characterization of a Novel Type of Acyltransferase

    PubMed Central

    Stöveken, Tim; Kalscheuer, Rainer; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

    2005-01-01

    The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria. PMID:15687201

  15. 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. PMID:26508171

  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. 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

  19. Acyl-coenzyme A:cholesterol acyltransferase 1 blockage enhances autophagy in the neurons of triple transgenic Alzheimer's disease mouse and reduces human P301L-tau content at the presymptomatic stage.

    PubMed

    Shibuya, Yohei; Niu, Zhaoyang; Bryleva, Elena Y; Harris, Brent T; Murphy, Stephanie R; Kheirollah, Alireza; Bowen, Zachary D; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Patients with Alzheimer's disease (AD) display amyloidopathy and tauopathy. In mouse models of AD, pharmacological inhibition using small molecule enzyme inhibitors or genetic inactivation of acyl-coenzyme A (Acyl-CoA):cholesterol acyltransferase 1 (ACAT1) diminished amyloidopathy and restored cognitive deficits. In microglia, ACAT1 blockage increases autophagosome formation and stimulates amyloid β peptide1-42 degradation. Here, we hypothesize that in neurons ACAT1 blockage augments autophagy and increases autophagy-mediated degradation of P301L-tau protein. We tested this possibility in murine neuroblastoma cells ectopically expressing human tau and in primary neurons isolated from triple transgenic AD mice that express mutant forms of amyloid precursor protein, presenilin-1, and human tau. The results show that ACAT1 blockage increases autophagosome formation and decreases P301L-tau protein content without affecting endogenous mouse tau protein content. In vivo, lacking Acat1 decreases P301L-tau protein content in the brains of young triple transgenic AD mice but not in those of old mice, where extensive hyperphosphorylations and aggregation of P301L-tau take place. These results suggest that, in addition to ameliorating amyloidopathy in both young and old AD mice, ACAT1 blockage may benefit AD by reducing tauopathy at early stage. PMID:25930235

  20. 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. PMID:24163338

  1. A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

    PubMed Central

    Carter, M E; Gulick, T; Moore, D D; Kelly, D P

    1994-01-01

    We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism. Images PMID:8007945

  2. LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters.

    PubMed

    Frey, Alexander J; Feldman, Daniel R; Trefely, Sophie; Worth, Andrew J; Basu, Sankha S; Snyder, Nathaniel W

    2016-05-01

    Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters. PMID:26968563

  3. 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 Central

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

    2015-01-01

    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

  4. Transgenic Arabidopsis flowers overexpressing acyl-CoA-binding protein ACBP6 are freezing tolerant.

    PubMed

    Liao, Pan; Chen, Qin-Fang; Chye, Mee-Len

    2014-06-01

    Low temperature stress adversely affects plant growth. It has been shown that the overexpression of ACYL-COENZYME A-BINDING PROTEIN6 (ACBP6) resulted in enhanced freezing tolerance in seedlings and rosettes accompanied by a decrease in phosphatidylcholine (PC), an increase in phosphatidic acid (PA) and an up-regulation of PHOSPHOLIPASE Dδ(PLDδ) in the absence of COLD-RESPONSIVE (COR)-related gene induction. Unlike rosettes, ACBP6-overexpressor (OE) flowers showed elevations in PC and monogalactosyldiacylglycerol (MGDG) accompanied by a decline in PA. The increase in PC species corresponded to a decline in specific PAs. To better understand such differences, the expression of PC-, MGDG-, proline-, ABA- and COR-related genes, and their transcription factors [C-repeat binding factors (CBFs), INDUCER OF CBF EXPRESSION1 (ICE1) and MYB15] was analyzed by quantitative real-time PCR (qRT-PCR). ACBP6-conferred freezing-tolerant flowers showed induction of COR-related genes, CBF genes and ICE1, PC-related genes (PLDδ, CK, CK-LIKE1, CK-LIKE2, CCT1, CCT2, LPCAT1, PLA2α, PAT-PLA-IIβ, PAT-PLA-IIIα, PAT-PLA-IIIδ and PLDζ2), MGDG-related genes (MGD genes and SFR2) and ABA-responsive genes. In contrast, ACBP6-conferred freezing-tolerant rosettes were down-regulated in COR-related genes, CBF1, PC-related genes (PEAMT1, PEAMT2, PEAMT3, CK1, CCT1, CCT2, PLA2α, PAT-PLA-IIIδ and PLDζ2), MGDG-related genes (MGD2, MGD3 and SFR2) and some ABA-responsive genes including KIN1 and KIN2. These results suggest that the mechanism in ACBP6-conferred freezing tolerance varies in different organs. PMID:24556610

  5. Clinical and genetical heterogeneity of late-onset multiple acyl-coenzyme A dehydrogenase deficiency

    PubMed Central

    2014-01-01

    Background Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder caused by deficiency of electron transfer flavoprotein or electron transfer flavoprotein dehydrogenase. The clinical picture of late-onset forms is highly variable with symptoms ranging from acute metabolic decompensations to chronic, mainly muscular problems or even asymptomatic cases. Methods All 350 cases of late-onset MADD reported in the literature to date have been analyzed and evaluated with respect to age at presentation, diagnostic delay, biochemical features and diagnostic parameters as well as response to treatment. Results Mean age at onset was 19.2 years. The mean delay between onset of symptoms and diagnosis was 3.9 years. Chronic muscular symptoms were more than twice as common as acute metabolic decompensations (85% versus 33% of patients, respectively). 20% had both acute and chronic symptoms. 5% of patients had died at a mean age of 5.8 years, while 3% of patients have remained asymptomatic until a maximum age of 14 years. Diagnosis may be difficult as a relevant number of patients do not display typical biochemical patterns of urine organic acids and blood acylcarnitines during times of wellbeing. The vast majority of patients carry mutations in the ETFDH gene (93%), while mutations in the ETFA (5%) and ETFB (2%) genes are the exceptions. Almost all patients with late-onset MADD (98%) are clearly responsive to riboflavin. Conclusions Late-onset MADD is probably an underdiagnosed disease and should be considered in all patients with acute or chronic muscular symptoms or acute metabolic decompensation with hypoglycemia, acidosis, encephalopathy and hepatopathy. This may not only prevent patients from invasive diagnostic procedures such as muscle biopsies, but also help to avoid fatal metabolic decompensations. PMID:25200064

  6. 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

  7. Hebrew as a Binding Force.

    ERIC Educational Resources Information Center

    Fischler, Ben-Zion

    1990-01-01

    The role of the Hebrew language as a cohesive force and the history of modern Hebrew instruction are chronicled. It is proposed that despite the scattering of its speakers and periods of use only as a literary or business language, Hebrew has been a binding force for the Jewish people. It was with considerable struggle that Hebrew gained…

  8. Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets[S

    PubMed Central

    Snyder, Nathaniel W.; Basu, Sankha S.; Worth, Andrew J.; Mesaros, Clementina; Blair, Ian A.

    2015-01-01

    Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [13C1]propionate and [13C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[2H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. PMID:25424005

  9. Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.

    PubMed

    Snyder, Nathaniel W; Basu, Sankha S; Worth, Andrew J; Mesaros, Clementina; Blair, Ian A

    2015-01-01

    Metabolism of propionate involves the activated acyl-thioester propionyl-CoA intermediate. We employed LC-MS/MS, LC-selected reaction monitoring/MS, and LC-high-resolution MS to investigate metabolism of propionate to acyl-CoA intermediates. We discovered that propionyl-CoA can serve as a precursor to the direct formation of a new six-carbon mono-unsaturated acyl-CoA. Time course and dose-response studies in human hepatocellular carcinoma HepG2 cells demonstrated that the six-carbon mono-unsaturated acyl-CoA was propionate-dependent and underwent further metabolism over time. Studies utilizing [(13)C1]propionate and [(13)C3]propionate suggested a mechanism of fatty acid synthesis, which maintained all six-carbon atoms from two propionate molecules. Metabolism of 2,2-[(2)H2]propionate to the new six-carbon mono-unsaturated acyl-CoA resulted in the complete loss of two deuterium atoms, indicating modification at C2 of the propionyl moiety. Coelution experiments and isotopic tracer studies confirmed that the new acyl-CoA was trans-2-methyl-2-pentenoyl-CoA. Acyl-CoA profiles following treatment of HepG2 cells with mono-unsaturated six-carbon fatty acids also supported this conclusion. Similar results were obtained with human platelets, mouse hepatocellular carcinoma Hepa1c1c7 cells, human bronchoalveolar carcinoma H358 cells, and human colon adenocarcinoma LoVo cells. Interestingly, trans-2-methyl-2-pentenoyl-CoA corresponds to a previously described acylcarnitine tentatively described in patients with propionic and methylmalonic acidemia. We have proposed a mechanism for this metabolic route consistent with all of the above findings. PMID:25424005

  10. 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. PMID:22275303

  11. Enantioselective analysis of ketone bodies in patients with beta-ketothiolase deficiency, medium-chain acyl coenzyme A dehydrogenase deficiency and ketonemic vomiting.

    PubMed

    Heil, M; Podebrad, F; Prado, E; Beck, T; Mosand, A; Sewell, A C; Böhles, H; Lehnert, W

    2000-03-10

    Enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS) is a valuable tool for the differentiation of enantiomers from complex matrices when present in trace amounts. The separation of chiral compounds provides further information on the diagnosis of diseases, and on normal and abnormal biochemical pathways. The formation of the normal urinary metabolite 3-hydroxy-2-methylbutanoic acid (HMBA), excreted in abnormally high amounts in beta-ketothiolase deficiency, is not absolutely clarified. Metabolic pathways involving this metabolite are isoleucine catabolism, as well as presumably beta-oxidation of fatty acids and ketogenesis. The latter two pathways are distinguishable in their enantioselectivity. Enantioselective analysis gives further information on interfering metabolic pathways and the selectivity of the enzyme(s) forming HMBA. Different ratios of the stereoisomers of HMBA in control urine samples and patients with beta-ketothiolase deficiency were detected. Analogous to HMBA urinary 3-hydroxybutanoic acid (HBA) was investigated in several diseases. The formation of HBA and HMBA is expected to result from the same or similar metabolic pathways. Differences in the enantiomeric ratio of HMBA may originate from the enantioselectivity of different enzyme systems. PMID:10755375

  12. Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate

    PubMed Central

    Garland, P. B.; Shepherd, D.; Yates, D. W.

    1965-01-01

    1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50μμmoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by β-oxidation takes place without detectable accumulation of acyl-CoA intermediates. PMID:16749169

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

    PubMed Central

    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

    2015-01-01

    Background 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. Objectives We investigated the interaction between thyroid dysfunction and Acot expression in adipose tissues and livers of thyrotoxic and hypothyroid mice. Methods 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. Results 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. Conclusions 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. PMID:26601074

  14. Effect of carbon chain length in acyl coenzyme A on the efficiency of enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid.

    PubMed

    Furumochi, Sachie; Onoda, Tatsuya; Cho, Yuko; Fuwa, Haruhiko; Sasaki, Makoto; Yotsu-Yamashita, Mari; Konoki, Keiichi

    2016-07-01

    Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in various bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the microsomal fraction from the digestive gland of bivalves. We have been using LC-MS/MS to identify OA-transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and surfactants for solubilizing membrane proteins decrease the sensitivity of LC-MS/MS. The present study examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14 (tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reaction. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and containing either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The three functional units can be conjugated with fluorescent reagents and are applicable to the development of a novel assay for DTX3. PMID:27231127

  15. Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates.

    PubMed Central

    Staels, B; Vu-Dac, N; Kosykh, V A; Saladin, R; Fruchart, J C; Dallongeville, J; Auwerx, J

    1995-01-01

    Epidemiological and transgenic animal studies have implicated apo C-III as a major determinant of plasma triglyceride metabolism. Since fibrates are very efficient in lowering triglycerides, it was investigated whether fibrates regulate apo C-III gene expression. Different fibrates lowered rat liver apo C-III mRNA levels up to 90% in a dose- and time-dependent manner, whereas intestinal apo C-III mRNA remained constant. This decrease in liver apo C-III mRNA was rapid (1 d) and reversible, since it was restored to control levels within 1 wk after cessation of treatment. In addition, fenofibrate treatment abolished the developmental rise of hepatic apo C-III mRNA observed during the suckling-weaning period. Administration of fibrates to rats induced liver and intestinal expression of the acyl CoA oxidase gene, the rate-limiting enzyme for peroxisomal beta-oxidation of fatty acids. In primary cultures of rat and human hepatocytes, fenofibric acid lowered apo C-III mRNA in a time- and dose-dependent manner. This reduction in apo C-III mRNA levels was accompanied by a decreased secretion of apo C-III in the culture medium of human hepatocytes. In rat hepatocytes fenofibric acid induced acyl CoA oxidase gene expression, whereas acyl CoA oxidase mRNA remained unchanged in human hepatocytes. Nuclear run-on and transient transfection experiments of a reporter construct driven by the human apo C-III gene promoter indicated that fibrates downregulate apo C-III gene expression at the transcriptional level. In conclusion, these studies demonstrate that fibrates decrease rat and human liver apo C-III gene expression. In humans the mechanisms appears to be independent of the induction of peroxisomal enzymes. This downregulation of liver apo C-III gene expression by fibrates may contribute to the hypotriglyceridemic action of these drugs. Images PMID:7860752

  16. Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG.

    PubMed Central

    Fitzmaurice, A M; Kolattukudy, P E

    1997-01-01

    The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C10 to n-C18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein. PMID:9098059

  17. Role of fatty acyl coenzyme A oxidase in the efflux of oxidized glutathione from perfused livers of rats treated with the peroxisome proliferator nafenopin.

    PubMed

    Conway, J G; Neptun, D A; Garvey, L K; Popp, J A

    1987-09-15

    The diffusion of H2O2 into the cytoplasm from peroxisomes during high rates of peroxisomal beta oxidation of fatty acids was studied in perfused livers from rats treated with the hepatocarcinogenic peroxisome proliferator, nafenopin. Efflux of oxidized glutathione (GSSG) into the bile was used as a measure of increased H2O2 supply for cytoplasmic glutathione peroxidase. Male F-344 rats were given methylcellulose vehicle or nafenopin (80 mg/kg/day) by gavage for 5-8 days and livers perfused in situ with Krebs-Henseleit buffer containing 50 microM taurocholate and 0.75 g/100 ml albumin. In livers from fed, vehicle-treated or fed, nafenopin-treated rats basal rates of GSSG efflux were about 60 nmol/g/h. Subsequent infusion of 350 microM lauric acid, an excellent substrate for peroxisomal beta-oxidation, had no effect on GSSG efflux. To maximize fatty acid oxidation rats were fasted 16-20 h. In livers from fasted, nafenopin-treated rats the basal rate of GSSG efflux was 384 +/- 85 (SE) nmol/g/h (n = 8). Subsequent infusion of lauric acid increased the rate to 940 +/- 138 nmol/g/h. In livers from fasted, vehicle-treated rats lauric acid caused GSSG efflux to increase slightly from 104 +/- 14 to 286 +/- 37 nmol/g/h (n = 9). Efflux of reduced glutathione in bile was similar in livers from fasted, vehicle-treated (163 +/- 15 nmol/g/h) and fasted, nafenopin-treated rats (135 +/- 17 nmol/g/h) and decreased about 30% with lauric acid infusion. N-Octanoyl and oleoyl coenzyme A were excellent substrates for cyanide-insensitive NAD+ reduction in liver homogenates from fasted, nafenopin-treated rats whereas n-butyl, linoleoyl, and arachidonyl coenzyme A were poor substrates. Infusion of octanoate and oleate caused large increases in GSSG efflux from perfused livers from fasted, nafenopin-treated rats. In contrast, butyrate, linoleate, and arachidonate had no effect on GSSG efflux from livers from fasted, nafenopin-treated rats. Octanoate, oleate, linoleate, butyrate, and arachidonate had no effect on GSSG efflux from livers from fasted, vehicle-treated rats. Infusion of 2-bromooctanoate (600 microM) completely blocked lauric acid-induced increases in GSSG efflux and acetoacetate and beta-hydroxybutyrate production in livers from fasted, nafenopin-treated rats. Infusion of 1-3-bis(2-chloroethyl)-1-nitrosourea reduced glutathione reductase activity by 90% but did not alter lauric acid-induced increases in GSSG efflux or ketogenesis in livers from fasted, nafenopin-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3621175

  18. Design and synthesis of simple, yet potent and selective non-ring-A pyripyropene A-based inhibitors of acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2).

    PubMed

    Zhan, Yang; Zhang, Xiao-Wei; Xiong, Ying; Li, Bo-Liang; Nan, Fa-Jun

    2016-01-14

    A series of pyripyropene A-based compounds were designed and synthesized by opening the upper section of the A-ring, which significantly simplifies the structure and synthesis from commercially available starting materials. Representative compound (-)-3 exhibited potent activity against ACAT2 and greater selectivity for ACAT2 than for ACAT1. PMID:26584338

  19. Pleiotropic Effect of AccD5 and AccE5 Depletion in Acyl-Coenzyme A Carboxylase Activity and in Lipid Biosynthesis in Mycobacteria

    PubMed Central

    Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Cabruja, Matías; Bardou, Fabienne; Quémard, Annaïk; Gago, Gabriela; Gramajo, Hugo

    2014-01-01

    Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs. PMID:24950047

  20. 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

  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. PMID:27171439

  2. Effect of Mycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide

    PubMed Central

    Trajkovic, Vladimir; Singh, Gyanesh; Singh, Balwan; Singh, Sarman; Sharma, Pawan

    2002-01-01

    Secreted proteins of Mycobacterium tuberculosis are major targets of the specific immunity in tuberculosis and constitute promising candidates for the development of more efficient vaccines and diagnostic tests. We show here that M. tuberculosis-specific antigen 10 (MTSA-10, originally designated CFP-10) can bind to the surface of mouse J774 macrophage-like cells and stimulate the secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). MTSA-10 also synergized with gamma interferon (IFN-γ) for the induction of the microbicidal free radical nitric oxide (NO) in J774 cells, as well as in bone marrow-derived and peritoneal macrophages. On the other hand, pretreatment of J774 cells with MTSA-10 markedly reduced NO but not TNF-α or interleukin 10 (IL-10) release upon subsequent stimulation with lipopolysaccharide or the cell lysate of M. tuberculosis. The presence of IFN-γ during stimulation with M. tuberculosis lysate antagonized the desensitizing effect of MTSA-10 pretreatment on macrophage NO production. The activation of protein tyrosine kinases (PTK) and the serine/threonine kinases p38 MAPK and ERK was apparently required for MTSA-10 induction of TNF-α and NO release, as revealed by specific kinase inhibitors. However, only p38 MAPK activity, not PTK or ERK activity, was partly responsible for MTSA-10-mediated macrophage desensitization. The modulation of macrophage function by MTSA-10 suggests a novel mechanism for its involvement in immunopathogenesis of tuberculosis and might have implications for the prevention, diagnosis, and therapy of this disease. PMID:12438325

  3. Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

    PubMed

    Pasternack, M S; Bleier, K J; McInerney, T N

    1991-08-01

    The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells. PMID:1860869

  4. 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.

  5. Human testis expresses a specific poly(A)-binding protein.

    PubMed

    Féral, C; Guellaën, G; Pawlak, A

    2001-05-01

    In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids. PMID:11328870

  6. Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-01-01

    Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid β-oxidation and phloem-mediated lipid transport is highlighted. PMID:27023243

  7. Plant Cytosolic Acyl-CoA-Binding Proteins.

    PubMed

    Ye, Zi-Wei; Chye, Mee-Len

    2016-01-01

    A gene family encoding six members of acyl-CoA-binding proteins (ACBP) exists in Arabidopsis and they are designated as AtACBP1-AtACBP6. They have been observed to play pivotal roles in plant lipid metabolism, consistent to the abilities of recombinant AtACBP in binding different medium- and long-chain acyl-CoA esters in vitro. While AtACBP1 and AtACBP2 are membrane-associated proteins with ankyrin repeats and AtACBP3 contains a signaling peptide for targeting to the apoplast, AtACBP4, AtACBP5 and AtACBP6 represent the cytosolic forms in the AtACBP family. They were verified to be subcellularly localized in the cytosol using diverse experimental methods, including cell fractionation followed by western blot analysis, immunoelectron microscopy and confocal laser-scanning microscopy using autofluorescence-tagged fusions. AtACBP4 (73.2 kDa) and AtACBP5 (70.1 kDa) are the largest, while AtACBP6 (10.4 kDa) is the smallest. Their binding affinities to oleoyl-CoA esters suggested that they can potentially transfer oleoyl-CoA esters from the plastids to the endoplasmic reticulum, facilitating the subsequent biosynthesis of non-plastidial membrane lipids in Arabidopsis. Recent studies on ACBP, extended from a dicot (Arabidopsis) to a monocot, revealed that six ACBP are also encoded in rice (Oryza sativa). Interestingly, three small rice ACBP (OsACBP1, OsACBP2 and OsACBP3) are present in the cytosol in comparison to one (AtACBP6) in Arabidopsis. In this review, the combinatory and distinct roles of the cytosolic AtACBP are discussed, including their functions in pollen and seed development, light-dependent regulation and substrate affinities to acyl-CoA esters. PMID:26662549

  8. 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

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

    PubMed

    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

  10. Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6

    PubMed Central

    Demangel, Caroline; Brodin, Priscille; Cockle, Paul J.; Brosch, Roland; Majlessi, Laleh; Leclerc, Claude; Cole, Stewart T.

    2004-01-01

    The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6. PMID:15039340

  11. 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

  12. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 5 2010-10-01 2010-10-01 false How must I provide a binding estimate? 375.403... TRANSPORTATION OF HOUSEHOLD GOODS IN INTERSTATE COMMERCE; CONSUMER PROTECTION REGULATIONS Estimating Charges § 375.403 How must I provide a binding estimate? (a) You may provide a guaranteed binding estimate...

  13. 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

  14. Investigation on the conA binding properties of Klebsiella pneumoniae.

    PubMed

    Anuar, A S S; Tay, S T

    2014-12-01

    Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation. PMID:25776607

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

    PubMed

    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

  16. Structure of armadillo ACBP: a new member of the acyl-CoA-binding protein family

    SciTech Connect

    Costabel, Marcelo D.; Ermácora, Mario R.; Santomé, José A.; Alzari, Pedro M.; Guérin, Diego M. A.

    2006-10-01

    The X-ray structure of the tetragonal form of apo acyl-CoA-binding protein (ACBP) from the Harderian gland of the South American armadillo Chaetophractus villosus has been solved. The X-ray structure of the tetragonal form of apo acyl-CoA-binding protein (ACBP) from the Harderian gland of the South American armadillo Chaetophractus villosus has been solved. ACBP is a carrier for activated long-chain fatty acids and has been associated with many aspects of lipid metabolism. Its secondary structure is highly similar to that of the corresponding form of bovine ACBP and exhibits the unique flattened α-helical bundle (up–down–down–up) motif reported for animal, yeast and insect ACBPs. Conformational differences are located in loops and turns, although these structural differences do not suffice to account for features that could be related to the unusual biochemistry and lipid metabolism of the Harderian gland.

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

    PubMed

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

    2013-10-11

    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. PMID:24041693

  18. 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.

  19. An integrated model for the nucleo-cytoplasmic transport of cytoplasmic poly(A)-binding proteins.

    PubMed

    Burgess, Hannah M; Gray, Nicola K

    2012-05-01

    Cytoplasmic poly(A)-binding proteins (PABPs) regulate mRNA stability and translation. Although predominantly localized in the cytoplasm, PABP proteins also cycle through the nucleus. Recent work has established that their steady-state localization can be altered by cellular stresses such as ultraviolet (UV) radiation, and infection by several viruses, resulting in nuclear accumulation of PABPs. Here, we present further evidence that their interaction with and release from mRNA and translation complexes are important in determining their sub-cellular distribution and propose an integrated model for regulated nucleo-cytoplasmic transport of PABPs. PMID:22896784

  20. 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.

  1. 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

  2. Cooperation of Escherichia coli Hfq hexamers in DsrA binding

    PubMed Central

    Wang, Weiwei; Wang, Lijun; Zou, Yang; Zhang, Jiahai; Gong, Qingguo; Wu, Jihui; Shi, Yunyu

    2011-01-01

    Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA and target mRNA. Hfq mediates DsrA-dependent translational activation of rpoS mRNA at low temperatures. rpoS encodes the stationary-phase σ factor σS, which is the central regulator in general stress response. However, structural information on Hfq–DsrA interaction is not yet available. Although Hfq is reported to hydrolyze ATP, the ATP-binding site is still unknown. Here, we report a ternary crystal complex structure of Escherichia coli Hfq bound to a major Hfq recognition region on DsrA (AU6A) together with ADP, and a crystal complex structure of Hfq bound to ADP. AU6A binds to the proximal and distal sides of two Hfq hexamers. ADP binds to a purine-selective site on the distal side and contacts conserved arginine or glutamine residues on the proximal side of another hexamer. This binding mode is different from previously postulated. The cooperation of two different Hfq hexamers upon nucleic acid binding in solution is verified by fluorescence polarization and solution nuclear magnetic resonance (NMR) experiments using fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with full-length Hfq and DsrA also supports cooperation of Hfq hexamers upon DsrA binding. The implications of Hfq hexamer cooperation have been discussed. PMID:21979921

  3. 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.

  4. Poly(A)-binding proteins and mRNA localization: who rules the roost?

    PubMed

    Gray, Nicola K; Hrabálková, Lenka; Scanlon, Jessica P; Smith, Richard W P

    2015-12-01

    RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. PMID:26614673

  5. Poly(A)-binding proteins are required for diverse biological processes in metazoans.

    PubMed

    Smith, Richard W P; Blee, Tajekesa K P; Gray, Nicola K

    2014-08-01

    PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area. PMID:25110030

  6. Embryonic poly(A)-binding protein (ePAB) phosphorylation is required for Xenopus oocyte maturation.

    PubMed

    Friend, Kyle; Brook, Matthew; Bezirci, F Betül; Sheets, Michael D; Gray, Nicola K; Seli, Emre

    2012-07-01

    Oocyte maturation and early embryonic development require the cytoplasmic polyadenylation and concomitant translational activation of stored maternal mRNAs. ePAB [embryonic poly(A)-binding protein, also known as ePABP and PABPc1-like] is a multifunctional post-transcriptional regulator that binds to poly(A) tails. In the present study we find that ePAB is a dynamically modified phosphoprotein in Xenopus laevis oocytes and show by mutation that phosphorylation at a four residue cluster is required for oocyte maturation. We further demonstrate that these phosphorylations are critical for cytoplasmic polyadenylation, but not for ePAB's inherent ability to promote translation. Our results provide the first insight into the role of post-translational modifications in regulating PABP protein activity in vivo. PMID:22497250

  7. Fibronectin is a binding partner for the myelin-associated glycoprotein (siglec-4a).

    PubMed

    Strenge, K; Brossmer, R; Ihrig, P; Schauer, R; Kelm, S

    2001-06-22

    The myelin-associated glycoprotein (MAG) mediates cell-cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG-Fc from NG108-15 cells and by microsequencing of two peptides derived from a 210-kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N(2)A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development. PMID:11423128

  8. Poly(A)-binding proteins are required for diverse biological processes in metazoans

    PubMed Central

    Smith, Richard W.P.; Blee, Tajekesa K.P.; Gray, Nicola K.

    2014-01-01

    PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area. PMID:25110030

  9. Purification and characterization of variants of acyl-CoA-binding protein in the bovine liver.

    PubMed Central

    Jensen, M S; Højrup, P; Rasmussen, J T; Knudsen, J

    1992-01-01

    Four differently modified forms of acyl-CoA-binding protein (ACBP) were identified in ACBP purified from bovine liver. The majority of the purified ACBP was focused at pH 5.9 in isoelectric focusing and could be shown to be N-acetylated ACBP without any further modifications. Two minor peaks were focused at pH 5.25 and 4.85 respectively. Mass spectrometry and sequence determination showed that the pI 5.25 form was acetylated at Lys18 and that the pI 4.85 form was malonylated in the same position. Furthermore, it could be shown that non-enzymic glycosylation occurred during purification. The acetylated and malonylated variants of ACBP were only found in adult cattle. Images Fig. 5. PMID:1622397

  10. Poly(A) RNA and Paip2 act as allosteric regulators of poly(A)-binding protein.

    PubMed

    Lee, Seung Hwan; Oh, Jungsic; Park, Jonghyun; Paek, Ki Young; Rho, Sangchul; Jang, Sung Key; Lee, Jong-Bong

    2014-02-01

    When bound to the 3' poly(A) tail of mRNA, poly(A)-binding protein (PABP) modulates mRNA translation and stability through its association with various proteins. By visualizing individual PABP molecules in real time, we found that PABP, containing four RNA recognition motifs (RRMs), adopts a conformation on poly(A) binding in which RRM1 is in proximity to RRM4. This conformational change is due to the bending of the region between RRM2 and RRM3. PABP-interacting protein 2 actively disrupts the bent structure of PABP to the extended structure, resulting in the inhibition of PABP-poly(A) binding. These results suggest that the changes in the configuration of PABP induced by interactions with various effector molecules, such as poly(A) and PABP-interacting protein 2, play pivotal roles in its function. PMID:24293655

  11. Cytoplasmic poly(A) binding protein C4 serves a critical role in erythroid differentiation.

    PubMed

    Kini, Hemant K; Kong, Jian; Liebhaber, Stephen A

    2014-04-01

    The expression of an mRNA is strongly impacted by its 3' poly(A) tail and associated poly(A)-binding proteins (PABPs). Vertebrates encode six PABP isoforms that vary in abundance, distribution, developmental control, and subcellular localization. Here we demonstrate that the minor PABP isoform PABPC4 is expressed in erythroid cells and impacts the steady-state expression of a subset of erythroid mRNAs. Motif analyses reveal a high-value AU-rich motif in the 3' untranslated regions (UTRs) of PABPC4-impacted mRNAs. This motif enhances the association of PABPC4 with mRNAs containing critically shortened poly(A) tails. This association may serve to protect a subset of mRNAs from accelerated decay. Finally, we demonstrate that selective depletion of PABPC4 in an erythroblast cell line inhibits terminal erythroid maturation with corresponding alterations in the erythroid gene expression. These observations lead us to conclude that PABPC4 plays an essential role in posttranscriptional control of a major developmental pathway. PMID:24469397

  12. Control of synaptic plasticity and memory via suppression of poly(A)-binding protein.

    PubMed

    Khoutorsky, Arkady; Yanagiya, Akiko; Gkogkas, Christos G; Fabian, Marc R; Prager-Khoutorsky, Masha; Cao, Ruifeng; Gamache, Karine; Bouthiette, Frederic; Parsyan, Armen; Sorge, Robert E; Mogil, Jeffrey S; Nader, Karim; Lacaille, Jean-Claude; Sonenberg, Nahum

    2013-04-24

    Control of protein synthesis is critical for synaptic plasticity and memory formation. However, the molecular mechanisms linking neuronal activity to activation of mRNA translation are not fully understood. Here, we report that the translational repressor poly(A)-binding protein (PABP)-interacting protein 2A (PAIP2A), an inhibitor of PABP, is rapidly proteolyzed by calpains in stimulated neurons and following training for contextual memory. Paip2a knockout mice exhibit a lowered threshold for the induction of sustained long-term potentiation and an enhancement of long-term memory after weak training. Translation of CaMKIIα mRNA is enhanced in Paip2a⁻/⁻ slices upon tetanic stimulation and in the hippocampus of Paip2a⁻/⁻ mice following contextual fear learning. We demonstrate that activity-dependent degradation of PAIP2A relieves translational inhibition of memory-related genes through PABP reactivation and conclude that PAIP2A is a pivotal translational regulator of synaptic plasticity and memory. PMID:23622065

  13. Poly(A)-binding proteins: structure, domain organization, and activity regulation.

    PubMed

    Eliseeva, I A; Lyabin, D N; Ovchinnikov, L P

    2013-12-01

    RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA interactions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed. PMID:24490729

  14. Polyalanine-independent Conformational Conversion of Nuclear Poly(A)-binding Protein 1 (PABPN1)*

    PubMed Central

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

    2012-01-01

    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. PMID:22570486

  15. Isolation and detection of human IgA using a streptococcal IgA-binding peptide.

    PubMed

    Sandin, Charlotta; Linse, Sara; Areschoug, Thomas; Woof, Jenny M; Reinholdt, Jesper; Lindahl, Gunnar

    2002-08-01

    Bacterial proteins that bind to the Fc part of IgG have found widespread use in immunology. A similar protein suitable for the isolation and detection of human IgA has not been described. Here, we show that a 50-residue synthetic peptide, designated streptococcal IgA-binding peptide (Sap) and derived from a streptococcal M protein, can be used for single-step affinity purification of human IgA. High affinity binding of IgA required the presence in Sap of a C-terminal cysteine residue, not present in the intact M protein. Passage of human serum through a Sap column caused depletion of >99% of the IgA, and elution of the column allowed quantitative recovery of highly purified IgA, for which the proportions of the IgA1 and IgA2 subclasses were the same as in whole serum. Moreover, immobilized Sap could be used for single-step purification of secretory IgA of both subclasses from human saliva, with a recovery of approximately 45%. The Sap peptide could also be used to specifically detect IgA bound to Ag. Together, these data indicate that Sap is a versatile Fc-binding reagent that may open new possibilities for the characterization of human IgA. PMID:12133959

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

    PubMed Central

    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

    2012-01-01

    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. PMID:22328160

  17. Concanavalin A binding glycoproteins in subcellular fractions from the developing rat cerebral cortex.

    PubMed

    Rudge, J S; Murphy, S

    1984-09-01

    Synaptic plasma membrane (SPM) and mitochondrial fractions were prepared from 3-50-day rat cerebral cortex and their purity assessed. The fractions were subjected to electrophoresis on slab gels, stained for protein, and overlaid with 125I-concanavalin A (ConA). ConA binding glycoproteins (CABGs) were revealed by autoradiography. In the SPM fraction CABGs of MW 25,000, 63,000, 80,000, 115,000, 174,000, and 239,000 increased while those of MW 47,000, 75,000, and 190,000 decreased developmentally. In the mitochondrial fraction, CABGs of MW 25,000, 44,000, 115,000 and 174,000 increased while those of 34,000, 43,000, 47,000, 51,000, 80,000, 107,000, and 195,000 decreased developmentally. CABGs of MW 32,000, 63,000, 88,000, 153,000, 190,000, and 239,000 appear to be unique to the SPM fraction and those of MW 34,000, 107,000, and 195,000 are unique to the mitochondrial fraction. PMID:6747641

  18. The human nuclear poly(a)-binding protein promotes RNA hyperadenylation and decay.

    PubMed

    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

  19. 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

    2016-01-01

    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. Cytoplasmic Poly(A) Binding Protein C4 Serves a Critical Role in Erythroid Differentiation

    PubMed Central

    Kini, Hemant K.; Kong, Jian

    2014-01-01

    The expression of an mRNA is strongly impacted by its 3′ poly(A) tail and associated poly(A)-binding proteins (PABPs). Vertebrates encode six PABP isoforms that vary in abundance, distribution, developmental control, and subcellular localization. Here we demonstrate that the minor PABP isoform PABPC4 is expressed in erythroid cells and impacts the steady-state expression of a subset of erythroid mRNAs. Motif analyses reveal a high-value AU-rich motif in the 3′ untranslated regions (UTRs) of PABPC4-impacted mRNAs. This motif enhances the association of PABPC4 with mRNAs containing critically shortened poly(A) tails. This association may serve to protect a subset of mRNAs from accelerated decay. Finally, we demonstrate that selective depletion of PABPC4 in an erythroblast cell line inhibits terminal erythroid maturation with corresponding alterations in the erythroid gene expression. These observations lead us to conclude that PABPC4 plays an essential role in posttranscriptional control of a major developmental pathway. PMID:24469397

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

    PubMed

    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

    2012-10-01

    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

  2. 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

    2016-03-01

    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.

  3. The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

    PubMed Central

    Reuter, L. Maximilian; Meinel, Dominik M.; Sträßer, Katja

    2015-01-01

    RNA polymerase III (RNAPIII) synthesizes most small RNAs, the most prominent being tRNAs. Although the basic mechanism of RNAPIII transcription is well understood, recent evidence suggests that additional proteins play a role in RNAPIII transcription. Here, we discovered by a genome-wide approach that Nab2, a poly(A)-binding protein important for correct poly(A) tail length and nuclear mRNA export, is present at all RNAPIII transcribed genes. The occupancy of Nab2 at RNAPIII transcribed genes is dependent on transcription. Using a novel temperature-sensitive allele of NAB2, nab2-34, we show that Nab2 is required for the occupancy of RNAPIII and TFIIIB at target genes. Furthermore, Nab2 interacts with RNAPIII, TFIIIB, and RNAPIII transcripts. Importantly, impairment of Nab2 function causes an RNAPIII transcription defect in vivo and in vitro. Taken together, we establish Nab2, an important mRNA biogenesis factor, as a novel player required for RNAPIII transcription by stabilizing TFIIIB and RNAPIII at promoters. PMID:26220998

  4. Arabidopsis ACBP6 is an acyl-CoA-binding protein associated with phospholipid metabolism

    PubMed Central

    Chen, Qin-Fang; Xiao, Shi

    2008-01-01

    In our recent paper in Plant Physiology, we showed that the Arabidopsis thaliana 10-kD acyl-CoA-binding protein, ACBP6, is subcellularly localized to the cytosol and that the overexpression of ACBP6 in transgenic Arabidopsis enhanced freezing tolerance. ACBP6-conferred freezing tolerance was independent of induced cold-regulated (COLD-RESPONSIVE) gene expression, but was correlated to an enhanced expression of phospholipase Dδ (PLDδ). Lipid analyses on cold-acclimated freezing-treated ACBP6-overexpressors revealed a decline in phosphatidylcholine (PC) and an elevation of phosphatidic acid (PA) in comparison to wild type. Furthermore, the His-tagged ACBP6 recombinant protein was observed using in vitro filter-binding assays to bind PC, but not PA or lysophosphatidylcholine. Taken together, our results implicate roles for ACBP6 in phospholipid metabolism that is related to gene regulation and PC-binding/transfer. This represents the first report demonstrating the in vitro binding of an ACBP to a phospholipid. The effect of ACBP6 on PLDδ expression is reminiscent of yeast 10-kD ACBP function in the regulation of genes associated with stress responses, fatty acid synthesis and phospholipid synthesis. However, the yeast ACBP regulates the expression of genes involved in phospholipid synthesis by donation of acyl-CoA esters and its binding to phospholipids remains to be demonstrated. PMID:19704440

  5. Deciphering the roles of acyl-CoA-binding proteins in plant cells.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-09-01

    Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed. PMID:26340904

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

    PubMed Central

    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. PMID:23060893

  7. 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. PMID:26676327

  8. Two-dimensional /sup 1/H NMR studies on cyclophilin, a cytosolic cyclosporin A binding protein

    SciTech Connect

    Dalgarno, D.C.; Harding, M.W.; Lazarides, A.; Handschumacher, R.E.; Armitage, I.M.

    1986-05-01

    Cyclophilin (CyP) is a specific cytosolic cyclosporin A (CsA) binding protein (163 residues) that has been implicated in the pharmacological action of this potent immunosuppressant. One and two-dimensional /sup 1/H NMR methods are being employed to elucidate the solution structural properties of CyP particularly as they relate to the binding site of CsA. The focal point for these studies is the single Trp (residue number120) in CyP which, in the 1:1 CyP:CsA complex (K/sub d/approx.2 x 10/sup -7/M), shows a 2 fold enhancement in its intrinsic fluorescence. Using 2D /sup 1/H NMR methods, a low resolution structure has been derived for a very hydrophobic domain containing the Trp residue using interresidue n.O.e. data between assigned spin systems and a distance geometry algorithm. The structure of this hydrophobic domain will be discussed in relation to the predicted ..cap alpha../..beta.. secondary structure of this protein and comparisons made between its structure in the drug free and complexed form of the protein.

  9. The Arabidopsis cytosolic Acyl-CoA-binding proteins play combinatory roles in pollen development.

    PubMed

    Hsiao, An-Shan; Yeung, Edward C; Ye, Zi-Wei; Chye, Mee-Len

    2015-02-01

    In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been demonstrated to function in plant stress responses and development. Three of these AtACBPs (AtACBP4-AtACBP6) are cytosolic proteins and all are expressed in floral organs as well as in other tissues. The roles of cytosolic AtACBPs in floral development were addressed in this study. To this end, a T-DNA insertional knockout mutant of acbp5 was characterized before use in crosses with the already available acbp4 and acbp6 T-DNA knockout mutants to examine their independent and combinatory functions in floral development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuole accumulation in the acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen was the most severe abnormality occurring in the double and triple mutants. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant, which also displayed reduced ability in in vitro pollen germination. Transgenic Arabidopsis expressing β-glucuronidase (GUS) driven from the various AtACBP promoters indicated that AtACBP6pro::GUS expression overlapped with AtACBP4pro::GUS expression in pollen grains and with AtACBP5pro::GUS expression in the microspores and tapetal cells. Taken together, these results suggest that the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development. PMID:25395473

  10. 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

  11. 14-3-3θ is a binding partner of rat Eag1 potassium channels.

    PubMed

    Hsu, Po-Hao; Miaw, Shi-Chuen; Chuang, Chau-Ching; Chang, Pei-Yu; Fu, Ssu-Ju; Jow, Guey-Mei; Chiu, Mei-Miao; Jeng, Chung-Jiuan

    2012-01-01

    The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons. PMID:22911758

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. The CD11c antigen couples concanavalin A binding to generation of superoxide anion in human phagocytes.

    PubMed Central

    Lacal, P M; Balsinde, J; Cabañas, C; Bernabeu, C; Sánchez-Madrid, F; Mollinedo, F

    1990-01-01

    We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils. Images Fig. 2. Fig. 3. PMID:1973035

  14. Dietary n-3 polyunsaturated fatty acids increase T-lymphocyte phospholipid mass and acyl-CoA binding protein expression.

    PubMed

    Collison, Lauren W; Collison, Robert E; Murphy, Eric J; Jolly, Christopher A

    2005-01-01

    Dietary flaxseed oil, which is enriched in alpha-linolenic acid, and fish oil, which is enriched in EPA and DHA, possess anti-inflammatory properties when compared with safflower oil, which is enriched in linoleic acid. The influence of flaxseed oil and fish oil feeding on lipid metabolism in T-lymphocytes is currently unknown. This study directly compared the effects of feeding safflower oil, flaxseed oil, and fish oil for 8 wk on splenic T-lymphocyte proliferation, phospholipid mass, and acyl-CoA binding protein expression in the rat. The data show that both flaxseed oil and fish oil increased acyl-CoA binding protein expression and phosphatidic acid mass in unstimulated T-lymphocytes when compared with safflower oil feeding. Fish oil feeding increased cardiolipin mass, whereas flaxseed oil had no effect. After stimulation, flaxseed oil and fish oil blunted T-lymphocyte interleukin-2 production and subsequent proliferation, which was associated with the lack of increased acyl-CoA binding protein expression. The results reported show evidence for a novel mechanism by which dietary flaxseed oil and fish oil suppress T-lymphocyte proliferation via changes in acyl-CoA binding protein expression and phospholipid mass. PMID:15825833

  15. Activation of the Klebsiella pneumoniae nifU promoter: identification of multiple and overlapping upstream NifA binding sites.

    PubMed Central

    Cannon, W V; Kreutzer, R; Kent, H M; Morett, E; Buck, M

    1990-01-01

    The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1. Images PMID:2186362

  16. The effects of down-regulating expression of Arabidopsis thaliana membrane-associated acyl-CoA binding protein 2 on acyl-lipid composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Multiple classes of acyl-CoA binding proteins are encoded by plant genomes, including a plant-unique class of predicted integral membrane-proteins. Transcript analysis revealed that both of the integral membrane-acyl-CoA binding proteins of Arabidopsis thaliana, ACBP1 and ACBP2, are expressed in al...

  17. Functional characterization of a fatty acyl-CoA binding protein (ACBP) from the apicomplexan Cryptosporidium parvum

    PubMed Central

    Zeng, Bin; Cai, Xiaomin; Zhu, Guan

    2006-01-01

    SUMMARY We have identified and conducted functional analysis of a fatty acyl-CoA binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum. The CpACBP1 gene encodes a protein of 268 aa that is 3X larger than the typical ACBP proteins (i.e., ∼90 aa) of humans and animals. Sequence analysis indicated that CpACBP1 consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankrin repeat sequence (∼170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 was palmitoyl-CoA. RT-PCR, Western blotting and immuno-labeling analyses clearly showed that the CpACBP1 gene was mainly expressed in the intracellular developmental stages and the level increases during the parasite development. Immunofluorescence microscopy shows that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in the lipid remodeling in the PVM or the transport of fatty acids across the membrane. PMID:16849800

  18. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    PubMed

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. PMID:26119729

  19. 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.

  20. A putative acyl-CoA-binding protein is a major phloem sap protein in rice (Oryza sativa L.).

    PubMed

    Suzui, Nobuo; Nakamura, Shin-ichi; Fujiwara, Toru; Hayashi, Hiroaki; Yoneyama, Tadakatsu

    2006-01-01

    The N-terminal amino-acid sequence of a major rice phloem-sap protein, named RPP10, was determined. RPP10 is encoded by a single gene in the rice genome. Its complete amino-acid sequence, predicted from the corresponding rice full-length cDNA, showed high similarity to plant acyl-CoA-binding proteins (ACBPs). Western blot analysis using anti-ACBP antiserum revealed that putative ACBP is abundant in the phloem sap of rice plants, and is also present in sieve-tube exudates of winter squash (Cucurbita maxima), oilseed rape (Brassica napus), and coconut palm (Cocos nucifera). These findings give rise to the idea that ACBP may involve lipid metabolism and regulation in the phloem. PMID:16804052

  1. 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

  2. Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

    PubMed Central

    Drawbridge, J; Grainger, J L; Winkler, M M

    1990-01-01

    Poly(A)-binding proteins (PABPs) are the best characterized messenger RNA-binding proteins of eucaryotic cells and have been identified in diverse organisms such as mammals and yeasts. The in vitro poly(A)-binding properties of these proteins have been studied intensively; however, little is known about their function in cells. In this report, we show that sea urchin eggs have two molecular weight forms of PABP (molecular weights of 66,000 and 80,000). Each of these has at least five posttranslationally modified forms. Both sea urchin PABPs are found in approximately 1:1 ratios in both cytoplasmic and nuclear fractions of embryonic cells. Quantification in eggs and embryos revealed that sea urchin PABPs are surprisingly abundant, composing about 0.6% of total cellular protein. This is 50 times more than required to bind all the poly(A) in the egg based on the binding stoichiometry of 1 PABP per 27 adenosine residues. We found that density gradient centrifugation strips PABP from poly(A) and therefore underestimates the amount of PABP complexed to poly(A)+ RNA in cell homogenates. However, large-pore gel filtration chromatography could be used to separate intact poly(A)-PABP complexes from free PABP. Using the gel filtration method, we found that the threefold increase in poly(A) content of the egg after fertilization is paralleled by an approximate fivefold increase in the amount of bound PABP. Furthermore, both translated and nontranslated poly(A)+ RNAs appear to be complexed to PABP. As expected from the observation that PABPs are so abundant, greater than 95% of the PABP of the cell is uncomplexed protein. Images PMID:2196442

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

    PubMed

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

    2012-08-15

    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

  4. 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

  5. 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. PMID:26492470

  6. 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.

  7. The Transformation Suppressor Pdcd4 Is a Novel Eukaryotic Translation Initiation Factor 4A Binding Protein That Inhibits Translation

    PubMed Central

    Yang, Hsin-Sheng; Jansen, Aaron P.; Komar, Anton A.; Zheng, Xiaojing; Merrick, William C.; Costes, Sylvain; Lockett, Stephen J.; Sonenberg, Nahum; Colburn, Nancy H.

    2003-01-01

    Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation. A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII. Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm. eIF4A is an ATP-dependent RNA helicase needed to unwind 5′ mRNA secondary structure. Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F. In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation. In contrast, Pdcd4D418A, a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation. Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039). In addition, both Pdcd4 and Pdcd4D418A bound to the middle region of eIF4G. The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G. Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1. PMID:12482958

  8. Poly(A) binding protein abundance regulates eukaryotic translation initiation factor 4F assembly in human cytomegalovirus-infected cells.

    PubMed

    McKinney, Caleb; Perez, Cesar; Mohr, Ian

    2012-04-10

    By commandeering cellular translation initiation factors, or destroying those dispensable for viral mRNA translation, viruses often suppress host protein synthesis. In contrast, cellular protein synthesis proceeds in human cytomegalovirus (HCMV)-infected cells, forcing viral and cellular mRNAs to compete for limiting translation initiation factors. Curiously, inactivating the host translational repressor 4E-BP1 in HCMV-infected cells stimulates synthesis of the cellular poly(A) binding protein (PABP), significantly increasing PABP abundance. Here, we establish that new PABP synthesis is translationally controlled by the HCMV-encoded UL38 mammalian target of rapamycin complex 1-activator. The 5' UTR within the mRNA encoding PABP contains a terminal oligopyrimidine (TOP) element found in mRNAs, the translation of which is stimulated in response to mitogenic, growth, and nutritional stimuli, and proteins encoded by TOP-containing mRNAs accumulated in HCMV-infected cells. Furthermore, UL38 expression was necessary and sufficient to regulate expression of a PABP TOP-containing reporter. Remarkably, preventing the rise in PABP abundance by RNAi impaired eIF4E binding to eIF4G, thereby reducing assembly of the multisubunit initiation factor eIF4F, viral protein production, and replication. This finding demonstrates that viruses can increase host translation initiation factor concentration to foster their replication and defines a unique mechanism whereby control of PABP abundance regulates eIF4F assembly. PMID:22431630

  9. The human IgA-Fc alpha receptor interaction and its blockade by streptococcal IgA-binding proteins.

    PubMed

    Woof, J M

    2002-08-01

    IgA plays a key role in immune defence of the mucosal surfaces. IgA can trigger elimination mechanisms against pathogens through the interaction of its Fc region with Fc alpha Rs (receptors specific for the Fc region of IgA) present on neutrophils, macrophages, monocytes and eosinophils. The human Fc alpha R (CD89) shares homology with receptors specific for the Fc region of IgG (Fc gamma Rs) and IgE (Fc epsilon RIs), but is a more distantly related member of the receptor family. CD89 interacts with residues lying at the interface of the two domains of IgA Fc, a site quite distinct from the homologous regions at the top of IgG and IgE Fc recognized by Fc gamma R and Fc epsilon RI respectively. Certain pathogenic bacteria express surface proteins that bind to human IgA Fc. Experiments with domain-swap antibodies and mutant IgAs indicate that binding of three such proteins (Sir22 and Arp4 of Streptococcus pyogenes and beta protein of group B streptococci) depend on sites in the Fc interdomain region of IgA, the binding region also used by CD89. Further, we have found that the streptococcal proteins can inhibit interaction of IgA with CD89, and have thereby identified a mechanism by which a bacterial IgA-binding protein may modulate IgA effector function. PMID:12196121

  10. 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

  11. Poly(A) Binding Protein 1 Enhances Cap-Independent Translation Initiation of Neurovirulence Factor from Avian Herpesvirus

    PubMed Central

    Tahiri-Alaoui, Abdessamad; Zhao, Yuguang; Sadigh, Yashar; Popplestone, James; Kgosana, Lydia; Smith, Lorraine P.; Nair, Venugopal

    2014-01-01

    Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek’s disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of ‘closed loop’ structure of mRNA. PMID:25503397

  12. A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast.

    PubMed Central

    Belostotsky, D A; Meagher, R B

    1996-01-01

    Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation. Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants. Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis. Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP. However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability. Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation. We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP. This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development. PMID:8776896

  13. 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. PMID:26938582

  14. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

    PubMed Central

    Tustian, Andrew D.; Endicott, Christine; Adams, Benjamin; Mattila, John; Bak, Hanne

    2016-01-01

    ABSTRACT There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process. PMID:26963837

  15. The binding versatility of plant acyl-CoA-binding proteins and their significance in lipid metabolism.

    PubMed

    Lung, Shiu-Cheung; Chye, Mee-Len

    2016-09-01

    Acyl-CoA esters are the activated form of fatty acids and play important roles in lipid metabolism and the regulation of cell functions. They are bound and transported by nonenzymic proteins such as the acyl-CoA-binding proteins (ACBPs). Although plant ACBPs were so named by virtue of amino acid homology to existing yeast and mammalian counterparts, recent studies revealed that ligand specificities of plant ACBPs are not restricted to acyl-CoA esters. Arabidopsis and rice ACBPs also interact with phospholipids, and their affinities to different acyl-CoA species and phospholipid classes vary amongst isoforms. Their ligands also include heavy metals. Interactors of plant ACBPs are further diversified due to the evolution of protein-protein interacting domains. This review summarizes our current understanding of plant ACBPs with a focus on their binding versatility. Their broad ligand range is of paramount significance in serving a multitude of functions during development and stress responses as discussed herein. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26747650

  16. Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

    PubMed

    Shanmugasundaram, T; Kumar, G K; Wood, H G

    1988-08-23

    Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway [Wood, H.G., Ragsdale, S.W., & Pezacka, E. (1986) FEMS Microbiol. Rev. 39, 345-362]. The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties. In this study, we have determined the role of tryptophans at these subsites. N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA. Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification. The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS. Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA. Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans. A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching. On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH. PMID:3219350

  17. 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

  18. EsiB, a Novel Pathogenic Escherichia coli Secretory Immunoglobulin A-Binding Protein Impairing Neutrophil Activation

    PubMed Central

    Pastorello, Ilaria; Rossi Paccani, Silvia; Rosini, Roberto; Mattera, Rossella; Ferrer Navarro, Mario; Urosev, Dunja; Nesta, Barbara; Lo Surdo, Paola; Del Vecchio, Mariangela; Rippa, Valentina; Bertoldi, Isabella; Gomes Moriel, Danilo; Laarman, Alexander J.; van Strijp, Jos A. G.; Daura, Xavier; Pizza, Mariagrazia; Serino, Laura; Soriani, Marco

    2013-01-01

    ABSTRACT In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site. PMID:23882011

  19. Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89.

    PubMed

    Pleass, R J; Areschoug, T; Lindahl, G; Woof, J M

    2001-03-16

    Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function. PMID:11096107

  20. Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively.

    PubMed

    Cheng, Shijun; Gallie, Daniel R

    2013-01-01

    The eukaryotic translation initiation factor (eIF) 4G functions as a scaffold protein that assembles components of the translation initiation complex required to recruit the 40S ribosomal subunit to an mRNA. Although many eukaryotes express two highly similar eIF4G isoforms, those in plants are highly divergent in size and sequence from one another and are referred to as eIF4G and eIFiso4G. Although the domain organization of eIFiso4G differs substantially from eIF4G orthologs in other species, the domain organization of plant eIF4G is largely unknown despite the fact that it is more similar in size and sequence to eIF4G of other eukaryotes. In this study, we show that eIF4G differs from eIFiso4G in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively. PMID:26824014

  1. 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

  2. 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

  3. 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. PMID:26013164

  4. 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. PMID:19107926

  5. 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

  6. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein

    PubMed Central

    Klima, Martin; Tóth, Dániel J.; Hexnerova, Rozalie; Baumlova, Adriana; Chalupska, Dominika; Tykvart, Jan; Rezabkova, Lenka; Sengupta, Nivedita; Man, Petr; Dubankova, Anna; Humpolickova, Jana; Nencka, Radim; Veverka, Vaclav; Balla, Tamas; Boura, Evzen

    2016-01-01

    Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. PMID:27009356

  7. 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).

  8. 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. PMID:25175022

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

    PubMed Central

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

    2015-01-01

    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. PMID:25755103

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

    PubMed

    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. PMID:26880548

  11. 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

  12. A Functional Variant at miR-520a Binding Site in PIK3CA Alters Susceptibility to Colorectal Cancer in a Chinese Han Population

    PubMed Central

    Ding, Lifang; Jiang, Zao; Chen, Qiaoyun; Qin, Rong; Fang, Yue

    2015-01-01

    An increasing body of evidence has indicated that polymorphisms in the miRNA binding site of target gene can alter the ability of miRNAs to bind their target genes and modulate the risk of cancer. We aimed to investigate the association between a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR and the risk of colorectal cancer (CRC) in a Chinese Han population. The polymorphism rs141178472 was analyzed in a case-control study, including 386 CRC patients and 394 age- and sex-matched controls; the relationship between the polymorphism and the risk of colorectal cancer was examined. Individuals carrying the rs141178472 CC genotype or C allele had an increased risk of developing CRC (CC versus TT, OR (95% CI): 1.716 (1.084–2.716), P = 0.022; C versus T, OR (95% CI): 1.258 (1.021–1.551), P = 0.033). Furthermore, the expression of PIK3CA was detected in the peripheral blood mononucleated cell of CRC patients, suggesting that mRNA levels of PIK3CA might be associated with SNP rs141178472. These findings provide evidence that a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR may play a role in the etiology of CRC. PMID:25834816

  13. 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.

  14. 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.

  15. EFIA/YB-1 is a component of cardiac HF-1A binding activity and positively regulates transcription of the myosin light-chain 2v gene.

    PubMed Central

    Zou, Y; Chien, K R

    1995-01-01

    Transient assays in cultured ventricular muscle cells and studies in transgenic mice have identified two adjacent regulatory elements (HF-1a and HF-1b/MEF-2) as required to maintain ventricular chamber-specific expression of the myosin light-chain 2v (MLC-2v) gene. A rat neonatal heart cDNA library was screened with an HF-1a binding site, resulting in the isolation of EFIA, the rat homolog of human YB-1. Purified recombinant EFIA/YB-1 protein binds to the HF-1a site in a sequence-specific manner and contacts a subset of the HF-1a contact points made by the cardiac nuclear factor(s). The HF-1a sequence contains AGTGG, which is highly homologous to the inverted CCAAT core of the EFIA/YB-1 binding sites and is found to be essential for binding of the recombinant EFIA/YB-1. Antiserum against Xenopus YB-3 (100% identical in the DNA binding domain and 89% identical in overall amino acid sequence to rat EFIA) can specifically abolish a component of the endogenous HF-1a complex in the rat cardiac myocyte nuclear extracts. In cotransfection assays, EFIA/YB-1 increased 250-bp MLC-2v promoter activity by 3.4-fold specifically in the cardiac cell context and in an HF-1a site-dependent manner. EFIA/YB-1 complexes with an unknown protein in cardiac myocyte nuclear extracts to form the endogenous HF-1a binding activity. Immunocoprecipitation revealed that EFIA/YB-1 has a major associated protein of approximately 30 kDa (p30) in cardiac muscle cells. This study suggests that EFIA/YB-1, together with the partner p30, binds to the HF-1a site and, in conjunction with HF-1b/MEF-2, mediates ventricular chamber-specific expression of the MLC-2v gene. PMID:7760795

  16. OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers.

    PubMed

    Zhao, Xinai; de Palma, Justina; Oane, Rowena; Gamuyao, Rico; Luo, Ming; Chaudhury, Abdul; Hervé, Philippe; Xue, Qingzhong; Bennett, John

    2008-05-01

    Hybrids lose heterotic yield advantage when multiplied sexually via meiosis. A potential alternative breeding system for hybrids is apospory, where female gametes develop without meiosis. Common among grasses, apospory begins in the nucellus, where aposporous initials (AIs) appear near the sexual megaspore mother cell (MeMC). The cellular origin of AIs is obscure, but one possibility, suggested by the mac1 and msp1 mutants of maize and rice, is that AIs are apomeiotic derivatives of the additional MeMCs that appear when genetic control over sporocyte numbers is relaxed. MULTIPLE SPOROCYTES1 (MSP1) encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. This phenotype is copied in mutants of TAPETUM DETERMINANT1 (TPD1), which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1. Here we show that rice contains two TPD1-like genes, OsTDL1A and OsTDL1B. Both are co-expressed with MSP1 in anthers during meiosis, but only OsTDL1A and MSP1 are co-expressed in the ovule. OsTDL1A binds to the leucine-rich-repeat domain of MSP1 in yeast two-hybrid assays and bimolecular fluorescence complementation in onion cells; OsTDL1B lacks this capacity. When driven by the maize Ubiquitin1 promoter, RNA interference against OsTDL1A phenocopies msp1 in the ovule but not in the anther. Thus, RNAi produces multiple MeMCs without causing male sterility. We conclude that OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice. PMID:18248596

  17. 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. PMID:25179842

  18. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer.

    PubMed

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  19. Site-specific cleavage of the host poly(A) binding protein by the encephalomyocarditis virus 3C proteinase stimulates viral replication.

    PubMed

    Kobayashi, Mariko; Arias, Carolina; Garabedian, Alexandra; Palmenberg, Ann C; Mohr, Ian

    2012-10-01

    Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication. PMID:22837200

  20. 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. PMID:25190455

  1. Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication.

    PubMed

    Wang, Xiaoye; Bai, Juan; Zhang, Lili; Wang, Xianwei; Li, Yufeng; Jiang, Ping

    2012-12-01

    Interactions between host factors and the viral protein play important roles in host adaptation and regulation of virus replication. Poly(A)-binding protein (PABP), a host cellular protein that enhances translational efficiency by circularizing mRNAs, was identified by yeast two-hybrid screening as a cellular partner for PRRSV nucleocapsid (N) protein in porcine alveolar macrophages. The specific interaction of PRRSV N protein with PABP was confirmed in infected cells by co-immunoprecipitation and in vitro by GST pull-down assay. We showed by confocal microscopy that the PABP co-localized with the PRRSV N protein. Using a series of deletion mutants, the interactive domain of N protein with PABP was mapped to a region of amino acids 52-69. For PABP, C-terminal half, which interestingly interacts other translation regulators, was determined to be the domain interactive with N protein. Short hairpin RNA (shRNA)-mediated silencing of PABP in cells resulted in significantly reduced PRRSV RNA synthesis, viral encoded protein expression and viral titer. Overall, the results presented here point toward an important role for PABP in regulating PRRSV replication. PMID:22985629

  2. A novel principle for conferring selectivity to poly(A)-binding proteins: interdependence of two ATP synthase beta-subunit mRNA-binding proteins.

    PubMed

    Andersson, U; Antonicka, H; Houstek, J; Cannon, B

    2000-02-15

    Based on electrophoretic mobility-shift assays and UV cross-linking experiments, we present evidence in the present work for the existence of two mammalian cytosolic proteins that selectively interact with the 3'-untranslated region of the mRNA coding for the catalytic beta-subunit of mitochondrial ATP synthase (beta-mtATPase). One of the proteins, beta-mtATPase mRNA-binding protein (BARB)1, is a novel poly(A)-binding protein that specifically binds the poly(A) tail of the beta-mtATPase transcript. BARB1 achieves this mRNA selectivity through its interaction with a second protein, BARB2, that binds the beta-mtATPase mRNA through a 22-bp element with a uridylate core, located 75 bp upstream of the poly(A) tail. Conversely, in the absence of BARB1, BARB2 is still able to bind the beta-mtATPase mRNA, but does so with lower affinity. Thus the interaction between BARB1 and BARB2 and beta-mtATPase mRNA involves the formation of a complex between the two BARB proteins. We conclude that BARB1 and BARB2 selectively bind the 3'-untranslated region of beta-mtATPase mRNA in a novel and interdependent manner. The complex between these two proteins may be involved in post-transcriptional regulation of gene expression. PMID:10657236

  3. Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans.

    PubMed

    Flamand, Mathieu N; Wu, Edlyn; Vashisht, Ajay; Jannot, Guillaume; Keiper, Brett D; Simard, Martin J; Wohlschlegel, James; Duchaine, Thomas F

    2016-07-01

    Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3' termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3'UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3'UTR poly(A) tail to the bound miRISC and deadenylase. PMID:27095199

  4. The relationship of rat liver overt carnitine palmitoyltransferase to the mitochondrial malonyl-CoA binding entity and to the latent palmitoyltransferase.

    PubMed Central

    Ghadiminejad, I; Saggerson, E D

    1990-01-01

    1. Confirming previous work [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPT1) from rat liver was found to be localized in outer rather than in inner mitochondrial membranes. 2. Antisera were raised against a liver mitochondrial CPT of Mr 68,000, which was presumed to be the latent from of the enzyme (CPT2). These antisera cross-reacted with solubilized CPT extracted from liver inner mitochondrial membranes and with polypeptides of Mr 68,000 and 60,000 in immunoblots of both inner and outer mitochondrial membranes. The antisera also precipitated CPT activity from detergent-treated total membrane and outer-membrane preparations. 3. The antisera did not precipitate [14C]malonyl-CoA binding material obtained either from total membranes or from outer membranes. 4. It was concluded that liver CPT1 and CPT2 have some epitopes in common and may have a similar subunit size. In addition, CPT1 and the entity that binds malonyl-CoA must be separated polypeptides. Images Fig. 2. Fig. 3. PMID:2241911

  5. Poly(A)-binding proteins are required for microRNA-mediated silencing and to promote target deadenylation in C. elegans

    PubMed Central

    Flamand, Mathieu N.; Wu, Edlyn; Vashisht, Ajay; Jannot, Guillaume; Keiper, Brett D.; Simard, Martin J.; Wohlschlegel, James; Duchaine, Thomas F.

    2016-01-01

    Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3′ termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3′UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3′UTR poly(A) tail to the bound miRISC and deadenylase. PMID:27095199

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

    PubMed Central

    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. PMID:25190455

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

    PubMed

    Wang, Qishan; Bag, Jnanankur

    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. PMID:16378590

  8. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer

    PubMed Central

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  9. 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. PMID:27095701

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

    PubMed Central

    Ray, Swagat; Anderson, Emma C.

    2016-01-01

    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. PMID:26936655

  11. A Single Mutation in the Glycophorin A Binding Site of Hepatitis A Virus Enhances Virus Clearance from the Blood and Results in a Lower Fitness Variant

    PubMed Central

    Costafreda, M. Isabel; Ribes, Enric; Franch, Àngels; Bosch, Albert

    2012-01-01

    Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site. PMID:22593170

  12. 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. PMID:24456391

  13. 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.

  14. 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

    2016-01-01

    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. PMID:26936655

  15. The role of the poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants.

    PubMed

    Gallie, Daniel R

    2014-09-01

    Translation initiation in eukaryotes requires the involvement of multiple initiation factors (eIFs) that facilitate the binding of the 40 S ribosomal subunit to an mRNA and assemble the 80 S ribosome at the correct initiation codon. eIF4F, composed of eIF4E, eIF4A, and eIF4G, binds to the 5'-cap structure of an mRNA and prepares an mRNA for recruitment of a 40 S subunit. eIF4B promotes the ATP-dependent RNA helicase activity of eIF4A and eIF4F needed to unwind secondary structure present in a 5'-leader that would otherwise impede scanning of the 40 S subunit during initiation. The poly(A) binding protein (PABP), which binds the poly(A) tail, interacts with eIF4G and eIF4B to promote circularization of an mRNA and stimulates translation by promoting 40 S subunit recruitment. Thus, these factors serve essential functions in the early steps of protein synthesis. Their assembly and function requires multiple interactions that are competitive in nature and determine the nature of interactions between the termini of an mRNA. In this review, the domain organization and partner protein interactions are presented for the factors in plants which share similarities with those in animals and yeast but differ in several important respects. The functional consequences of their interactions on factor activity are also discussed. PMID:26779409

  16. Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region.

    PubMed

    Iwakawa, Hiro-Oki; Tajima, Yuri; Taniguchi, Takako; Kaido, Masanori; Mise, Kazuyuki; Tomari, Yukihide; Taniguchi, Hisaaki; Okuno, Tetsuro

    2012-08-01

    Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA. PMID:22593149

  17. Post-transcriptional regulation of the human inducible nitric oxide synthase (iNOS) expression by the cytosolic poly(A)-binding protein (PABP).

    PubMed

    Casper, Ingrid; Nowag, Sebastian; Koch, Kathrin; Hubrich, Thomas; Bollmann, Franziska; Henke, Jenny; Schmitz, Katja; Kleinert, Hartmut; Pautz, Andrea

    2013-09-01

    Affinity purification using the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3'-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or β2-microglobuline (β2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and β2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5'-UTR and two different binding sites in the 3'-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5'-UTR but not in the 3'-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5'-UTR or iNOS-3'-UTR luciferase reporter constructs. In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5'-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression. PMID:23711718

  18. Control of translation and miRNA-dependent repression by a novel poly(A) binding protein, hnRNP-Q.

    PubMed

    Svitkin, Yuri V; Yanagiya, Akiko; Karetnikov, Alexey E; Alain, Tommy; Fabian, Marc R; Khoutorsky, Arkady; Perreault, Sandra; Topisirovic, Ivan; Sonenberg, Nahum

    2013-01-01

    Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3' poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5' m(7)G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs. PMID:23700384

  19. Nanopore detachment kinetics of poly(A) binding proteins from RNA molecules reveals the critical role of C-terminus interactions.

    PubMed

    Lin, Jianxun; Fabian, Marc; Sonenberg, Nahum; Meller, Amit

    2012-03-21

    The ubiquitous and abundant cytoplasmic poly(A) binding protein (PABP) is a highly conserved multifunctional protein, many copies of which bind to the poly(A) tail of eukaryotic mRNAs to promote translation initiation. The N-terminus of PABP is responsible for the high binding specificity and affinity to poly(A), whereas the C-terminus is known to stimulate PABP multimerization on poly(A). Here, we use single-molecule nanopore force spectroscopy to directly measure interactions between poly(A) and PABPs. Both electrical and biochemical results show that the C-C domain interaction between two consecutive PABPs promotes cooperative binding. Up to now, investigators have not been able to probe the detailed polarity configuration (i.e., the internal arrangement of two PABPs on a poly(A) streak in which the C-termini face toward or away from each other). Our nanopore force spectroscopy system is able to distinguish the cooperative binding conformation from the noncooperative one. The ∼50% cooperative binding conformation of wild-type PABPs indicates that the C-C domain interaction doubles the cooperative binding probability. Moreover, the longer dissociation time of a cooperatively bound poly(A)/PABP complex as compared with a noncooperatively bound one indicates that the cooperative mode is the most stable conformation for PABPs binding onto the poly(A). However, ∼50% of the poly(A)/PABP complexes exhibit a noncooperative binding conformation, which is in line with previous studies showing that the PABP C-terminal domain also interacts with additional protein cofactors. PMID:22455926

  20. Rotavirus variant replicates efficiently although encoding an aberrant NSP3 that fails to induce nuclear localization of poly(A)-binding protein.

    PubMed

    Arnold, Michelle M; Brownback, Catie Small; Taraporewala, Zenobia F; Patton, John T

    2012-07-01

    The rotavirus (RV) non-structural protein NSP3 forms a dimer that has binding domains for the translation initiation factor eIF4G and for a conserved 3'-terminal sequence of viral mRNAs. Through these activities, NSP3 has been proposed to promote viral mRNA translation by directing circularization of viral polysomes. In addition, by disrupting interactions between eIF4G and the poly(A)-binding protein (PABP), NSP3 has been suggested to inhibit translation of host polyadenylated mRNAs and to stimulate relocalization of PABP from the cytoplasm to the nucleus. Herein, we report the isolation and characterization of SA11-4Fg7re, an SA11-4F RV derivative that contains a large sequence duplication initiating within the genome segment (gene 7) encoding NSP3. Our analysis showed that mutant NSP3 (NSP3m) encoded by SA11-4Fg7re is almost twice the size of the wild-type protein and retains the capacity to dimerize. However, in comparison to wild-type NSP3, NSP3m has a decreased capacity to interact with eIF4G and to suppress the translation of polyadenylated mRNAs. In addition, NSP3m fails to induce the nuclear accumulation of PABP in infected cells. Despite the defective activities of NSP3m, the levels of viral protein and progeny virus produced in SA11-4Fg7re- and SA11-4F-infected cells were indistinguishable. Collectively, these data are consistent with a role for NSP3 in suppressing host protein synthesis through antagonism of PABP activity, but also suggest that NSP3 functions may have little or no impact on the efficiency of virus replication in widely used RV-permissive cell lines. PMID:22442114

  1. Single nucleotide polymorphism in the microRNA-199a binding site of HIF1A gene is associated with pancreatic ductal adenocarcinoma risk and worse clinical outcomes

    PubMed Central

    Zhao, Tiansuo; Ma, Weidong; Dong, Jie; Zhang, Shengjie; Xin, Wen; Yang, Shengyu; Jia, Li; Hao, Jihui

    2016-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) is over-expressed in many cancers including pancreatic ductal adenocarcinoma (PDAC) and correlated with poor prognosis. We aim to determine the effect of germline genetic variants on the regulation of the homeostasis of the miRNA-gene regulatory loop in HIF1A gene and PDAC risk. HIF1A rs2057482 single nucleotide polymorphism (SNP) was genotyped in 410 PDAC cases and 490 healthy controls. The CC genotype SNP HIF1A is significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293–2.286) and shorter overall survival (OS, P<0.0001) compared with the CT/TT alleles group. The C/T variants of rs2057482, a SNP located near the miR-199a binding site in HIF1A, could lead to differential regulation of HIF1A by miR-199a. Specifically, the C allele of rs2057482 weakened miR-199a–induced repression of HIF-1α expression on both mRNA and protein levels. In the PDAC tissue, individuals with the rs2057482-CC genotype expressed significantly higher levels of HIF-1α protein than those with the rs2057482-CT/TT genotype (P<0.0001). Both the CC genotype of SNP HIF1A and increased HIF-1α expression are significantly associated with shorter OS of patients with PDAC. After adjusted by TNM staging, differentiation grade, and the levels of CA19-9, both SNP HIF1A and HIF-1α expression retained highly significance on OS (P<0.0001). Taken together, our study demonstrates that host genetic variants could disturb the regulation of the miR-199a/HIF1A regulatory loop and alter PDAC risk and poor prognosis. In conclusion, the rs2057482-CC genotype increases the susceptibility to PDAC and associated with cancer progression. PMID:26872370

  2. 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

  3. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    PubMed

    Park, Richard; El-Guindy, Ayman; Heston, Lee; Lin, Su-Fang; Yu, Kuan-Ping; Nagy, Mate; Borah, Sumit; Delecluse, Henri-Jacques; Steitz, Joan; Miller, George

    2014-01-01

    Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors. PMID:24705134

  4. Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Chen, Qin-Fang; Sooriyaarachchi, Sanjeewani; Mowbray, Sherry L.; Napier, Johnathan A.; Tanner, Julian A.; Chye, Mee-Len

    2014-01-01

    Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds. PMID:25423293

  5. Regulation of poly(A) binding protein function in translation: Characterization of the Paip2 homolog, Paip2B

    PubMed Central

    Berlanga, Juan José; Baass, Alexis; Sonenberg, Nahum

    2006-01-01

    The 5′ cap and 3′ poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This synergy is mediated via interactions between eIF4G (a component of the eIF4F cap binding complex) and poly(A) binding protein (PABP). Paip2 (PABP-interacting protein 2) binds PABP and inhibits translation both in vitro and in vivo by decreasing the affinity of PABP for polyadenylated RNA. Here, we describe the functional characteristics of Paip2B, a Paip2 homolog. A full-length brain cDNA of Paip2B encodes a protein that shares 59% identity and 80% similarity with Paip2 (Paip2A), with the highest conservation in the two PABP binding domains. Paip2B acts in a manner similar to Paip2A to inhibit translation of capped and polyadenylated mRNAs both in vitro and in vivo by displacing PABP from the poly(A) tail. Also, similar to Paip2A, Paip2B does not affect the translation mediated by the internal ribosome entry site (IRES) of hepatitis C virus (HCV). However, Paip2A and Paip2B differ with respect to both mRNA and protein distribution in different tissues and cell lines. Paip2A is more highly ubiquitinated than is Paip2B and is degraded more rapidly by the proteasome. Paip2 protein degradation may constitute a primary mechanism by which cells regulate PABP activity in translation. PMID:16804161

  6. 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.

  7. Isolation and characterization of a humoral factor that stimulates transcription of the acyl-CoA-binding protein in the pheromone gland of the silkmoth, Bombyx mori.

    PubMed

    Ohnishi, Atsushi; Koshino, Hiroyuki; Takahashi, Shunya; Esumi, Yasuaki; Matsumoto, Shogo

    2005-02-11

    Acyl-CoA binding protein (ACBP) is a highly conserved 10-kDa intracellular lipid-binding protein that binds straight-chain (C14-C22) acyl-CoA esters with high affinity and is expressed in a wide variety of species ranging from yeast to mammals. Functionally, ACBP can act as an acyl-CoA carrier or as an acyl-CoA pool maker within the cell. Much work on the biochemical properties regarding the ACBP has been performed using various vertebrate and plant tissues, as well as different types of cells in culture, the regulatory mechanisms underlying ACBP gene expression have remained poorly understood. By exploiting the unique sex pheromone production system in the moth pheromone gland (PG), we report that transcription of a specific ACBP termed pheromone gland ACBP is triggered by a hemolymph-based humoral factor. Following purification and structure elucidation by means of high resolution electrospray ionization mass spectrometry and NMR analyses, in conjunction with stereochemical analyses using acid hydrolysates, the humoral factor was identified to be beta-D-glucosyl-O-L-tyrosine. Examination of the hemolymph titers during development revealed that the amount of beta-D-glucosyl-O-L-tyrosine dramatically rose prior to eclosion and reached a maximum of 5 mg/ml (about 1 mg/pupa) on the day preceding eclosion, which was consistent with the effective dose of beta-D-glucosyl-O-L-tyrosine in stimulating pheromone gland ACBP transcription in vivo. Furthermore, in vitro assays using trimmed PG indicated that beta-D-glucosyl-O-L-tyrosine acts directly on the PG. These results provide the first evidence that transcription of some ACBPs can be triggered by specific humoral factors. PMID:15590686

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

    PubMed

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

    2016-04-15

    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 byPPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn(2+)-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

  9. Novel anti-Cryptosporidium activity of known drugs identified by high-throughput screening against parasite fatty acyl-CoA binding protein (ACBP)

    PubMed Central

    Fritzler, Jason M.; Zhu, Guan

    2012-01-01

    Background Cryptosporidium parvum causes an opportunistic infection in AIDS patients, and no effective treatments are yet available. This parasite possesses a single fatty acyl-CoA binding protein (CpACBP1) that is localized to the unique parasitophorous vacuole membrane (PVM). The major goal of this study was to identify inhibitors from known drugs against CpACBP1 as potential new anti-Cryptosporidium agents. Methods A fluorescence assay was developed to detect CpACBP1 activity and to identify inhibitors by screening known drugs. Efficacies of top CpACBP1 inhibitors against Cryptosporidium growth in vitro were evaluated using a quantitative RT–PCR assay. Results Nitrobenzoxadiazole-labelled palmitoyl-CoA significantly increased the fluorescent emission upon binding to CpACBP1 (excitation/emission 460/538 nm), which was quantified to determine the CpACBP1 activity and binding kinetics. The fluorescence assay was used to screen a collection of 1040 compounds containing mostly known drugs, and identified the 28 most active compounds that could inhibit CpACBP1 activity with sub-micromolar IC50 values. Among them, four compounds displayed efficacies against parasite growth in vitro with low micromolar IC50 values. The effective compounds were broxyquinoline (IC50 64.9 μM), cloxyquin (IC50 25.1 μM), cloxacillin sodium (IC50 36.2 μM) and sodium dehydrocholate (IC50 53.2 μM). Conclusions The fluorescence ACBP assay can be effectively used to screen known drugs or other compound libraries. Novel anti-Cryptosporidium activity was observed in four top CpACBP1 inhibitors, which may be further investigated for their potential to be repurposed to treat cryptosporidiosis and to serve as leads for drug development. PMID:22167242

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

    PubMed Central

    Fatscher, Tobias; Boehm, Volker; Weiche, Benjamin

    2014-01-01

    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. PMID:25147240

  11. Differential Localization of the Two T. brucei Poly(A) Binding Proteins to the Nucleus and RNP Granules Suggests Binding to Distinct mRNA Pools

    PubMed Central

    Kramer, Susanne; Bannerman-Chukualim, Bridget; Ellis, Louise; Boulden, Elizabeth A.; Kelly, Steve; Field, Mark C.; Carrington, Mark

    2013-01-01

    The number of paralogs of proteins involved in translation initiation is larger in trypanosomes than in yeasts or many metazoan and includes two poly(A) binding proteins, PABP1 and PABP2, and four eIF4E variants. In many cases, the paralogs are individually essential and are thus unlikely to have redundant functions although, as yet, distinct functions of different isoforms have not been determined. Here, trypanosome PABP1 and PABP2 have been further characterised. PABP1 and PABP2 diverged subsequent to the differentiation of the Kinetoplastae lineage, supporting the existence of specific aspects of translation initiation regulation. PABP1 and PABP2 exhibit major differences in intracellular localization and distribution on polysome fractionation under various conditions that interfere with mRNA metabolism. Most striking are differences in localization to the four known types of inducible RNP granules. Moreover, only PABP2 but not PABP1 can accumulate in the nucleus. Taken together, these observations indicate that PABP1 and PABP2 likely associate with distinct populations of mRNAs. The differences in localization to inducible RNP granules also apply to paralogs of components of the eIF4F complex: eIF4E1 showed similar localization pattern to PABP2, whereas the localisation of eIF4E4 and eIF4G3 resembled that of PABP1. The grouping of translation initiation as either colocalizing with PABP1 or with PABP2 can be used to complement interaction studies to further define the translation initiation complexes in kinetoplastids. PMID:23382864

  12. 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

  13. 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

  14. 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.

  15. Single nucleotide polymorphism in the microRNA-199a binding site of HIF1A gene is associated with pancreatic ductal adenocarcinoma risk and worse clinical outcomes.

    PubMed

    Wang, Xiuchao; Ren, He; Zhao, Tiansuo; Ma, Weidong; Dong, Jie; Zhang, Shengjie; Xin, Wen; Yang, Shengyu; Jia, Li; Hao, Jihui

    2016-03-22

    Hypoxia-inducible factor-1 alpha (HIF-1α) is over-expressed in many cancers including pancreatic ductal adenocarcinoma (PDAC) and correlated with poor prognosis. We aim to determine the effect of germline genetic variants on the regulation of the homeostasis of the miRNA-gene regulatory loop in HIF1A gene and PDAC risk. HIF1A rs2057482 single nucleotide polymorphism (SNP) was genotyped in 410 PDAC cases and 490 healthy controls. The CC genotype SNP HIF1A is significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293-2.286) and shorter overall survival (OS, P<0.0001) compared with the CT/TT alleles group. The C/T variants of rs2057482, a SNP located near the miR-199a binding site in HIF1A, could lead to differential regulation of HIF1A by miR-199a. Specifically, the C allele of rs2057482 weakened miR-199a-induced repression of HIF-1α expression on both mRNA and protein levels. In the PDAC tissue, individuals with the rs2057482-CC genotype expressed significantly higher levels of HIF-1α protein than those with the rs2057482-CT/TT genotype (P<0.0001). Both the CC genotype of SNP HIF1A and increased HIF-1α expression are significantly associated with shorter OS of patients with PDAC. After adjusted by TNM staging, differentiation grade, and the levels of CA19-9, both SNP HIF1A and HIF-1α expression retained highly significance on OS (P<0.0001). Taken together, our study demonstrates that host genetic variants could disturb the regulation of the miR-199a/HIF1A regulatory loop and alter PDAC risk and poor prognosis. In conclusion, the rs2057482-CC genotype increases the susceptibility to PDAC and associated with cancer progression. PMID:26872370

  16. Poly(A)-Binding Protein Facilitates Translation of an Uncapped/Nonpolyadenylated Viral RNA by Binding to the 3′ Untranslated Region

    PubMed Central

    Iwakawa, Hiro-oki; Tajima, Yuri; Taniguchi, Takako; Kaido, Masanori; Mise, Kazuyuki; Tomari, Yukihide; Taniguchi, Hisaaki

    2012-01-01

    Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5′ cap and a 3′ poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3′ untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3′CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3′CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3′CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3′ UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3′CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3′CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3′ UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3′CITE as substitutes for the 3′ poly(A) tail and the 5′ cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA. PMID:22593149

  17. Isotype-specific immunoregulation; characterization and function of Fc receptors on T-T hybridomas which produce murine IgA-binding factor.

    PubMed

    Kurita, T; Kiyono, H; Komiyama, K; Grossi, C E; Mestecky, J; McGhee, J R

    1986-06-01

    Several methods have been used in the present study to characterize Fc receptors (FcR) expressed on T-T hybridomas derived from mouse Peyer's patch T helper (Th) cell clones that preferentially support IgA responses. These T hybridomas (designated Th HA cells) produce IgA-binding factor (IBF alpha) which regulates antigen-dependent IgA responses. The ultrastructure of Th HA cells and the distribution of Fc alpha R on these cell lines were determined by colloidal gold (CG) immunoelectron microscopy (IEM). When Th HA cells were incubated with purified mouse IgA followed by CG-labeled anti-IgA, an even pattern of CG was distributed on the cell membrane. To ensure that binding occurred through Fc alpha R, Th HA cells were mixed with MOPC 315 IgA anti-DNP, followed by staining with CG-labeled TNP-human serum albumin. This resulted in an identical pattern of gold particle distribution, confirming expression of Fc alpha R on Th HA cells. No Fc mu R or Fc gamma 1R were detectable on Th HA cells by IEM. Immunocytoadherence with TNP-conjugated erythrocytes confirmed that Th HA cells were Fc alpha R+; however, no IgM or IgG rosettes were seen. When these cell lines were analyzed by flow cytometry (FACS) using IgA, IgM, or IgG1 and FITC-labeled anti-H chain-specific antibodies, 55 to 65% of cultured Th HA cells expressed Fc alpha R, and 11 to 18% expressed Fc mu R; however, no Fc gamma 1R was detectable on Th HA cells. The use of ELISA with Th HA cells as antigen confirmed the expression of Fc alpha R and the presence of less Fc mu R on these two cell lines. Solubilized membrane fractions derived from Th HA cells were tested for the presence of FcR by ELISA and for biologic function for support of IgA responses in Peyer's patch B cell cultures. Both Fc alpha R and Fc mu R were detected in fractions derived from Th HA cells. Furthermore, these fractions supported in vitro IgA anti-sheep erythrocyte responses, comparable to those obtained with Th HA cell culture supernatants

  18. Phosphatidylinositol 4,5-biphosphate (PIP2) modulates syntaxin-1A binding to sulfonylurea receptor 2A to regulate cardiac ATP-sensitive potassium (KATP) channels.

    PubMed

    Xie, Li; Liang, Tao; Kang, Youhou; Lin, Xianguang; Sobbi, Roozbeh; Xie, Huanli; Chao, Christin; Backx, Peter; Feng, Zhong-Ping; Shyng, Show-Ling; Gaisano, Herbert Y

    2014-10-01

    Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cells showed that increasing endogenous or exogenous PIP2 diminished the inhibitory effect of Syn-1A on KATP currents. The physiological relevance of these findings was confirmed by ability of exogenous PIP2 to block exogenous Syn-1A inhibition of cardiac KATP currents in inside-out patches of mouse ventricular myocytes. The effect of PIP2 on physical and functional interactions between Syn-1A and KATP channels is specific and not observed with physiologic concentrations of other phospholipids. To unequivocally demonstrate the specificity of PIP2 interaction with Syn-1A and its impact on KATP channel modulation by Syn-1A, we employed a PIP2-insensitive Syn-1A-5RK/A mutant. The Syn-1A-5RK/A mutant retains the ability to interact with SUR2A in both in vitro binding and in vivo FRET assays, although as expected the interaction is no longer disrupted by PIP2. Interestingly, at physiological PIP2 concentrations, Syn-1A-5RK/A inhibited KATP currents to a greater extent than Syn-1A-WT, indicating

  19. 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

  20. 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…

  1. 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.

  2. 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.

  3. ACBD2/ECI2-Mediated Peroxisome-Mitochondria Interactions in Leydig Cell Steroid Biosynthesis.

    PubMed

    Fan, Jinjiang; Li, Xinlu; Issop, Leeyah; Culty, Martine; Papadopoulos, Vassilios

    2016-07-01

    Fatty acid metabolism and steroid biosynthesis are 2 major pathways shared by peroxisomes and mitochondria. Both organelles are in close apposition to the endoplasmic reticulum, with which they communicate via interorganelle membrane contact sites to promote cellular signaling and the exchange of ions and lipids. To date, no convincing evidence of the direct contact between peroxisomes and mitochondria was reported in mammalian cells. Hormone-induced, tightly controlled steroid hormone biosynthesis requires interorganelle interactions. Using immunofluorescent staining and live-cell imaging, we found that dibutyryl-cAMP treatment of MA-10 mouse tumor Leydig cells rapidly induces peroxisomes to approach mitochondria and form peroxisome-mitochondrial contact sites/fusion, revealed by the subcellular distribution of the endogenous acyl-coenzyme A-binding domain (ACBD)2/ECI2 isoform A generated by alternative splicing, and further validated using a proximity ligation assay. This event occurs likely via a peroxisome-like structure, which is mediated by peroxisomal and mitochondrial matrix protein import complexes: peroxisomal import receptor peroxisomal biogenesis factor 5 (PEX5), and the mitochondrial import receptor subunit translocase of outer mitochondrial membrane 20 homolog (yeast) protein. Similar results were obtained using the mLTC-1 mouse tumor Leydig cells. Ectopic expression of the ACBD2/ECI2 isoform A in MA-10 cells led to increased basal and hormone-stimulated steroid formation, indicating that ACBD2/ECI2-mediated peroxisomes-mitochondria interactions favor in the exchange of metabolites and/or macromolecules between these 2 organelles in support of steroid biosynthesis. Considering the widespread occurrence of the ACBD2/ECI2 protein, we propose that this protein might serve as a tool to assist in understanding the contact between peroxisomes and mitochondria. PMID:27167610

  4. 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

  5. ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.; DeRisi, Joseph L.

    2013-01-01

    ABSTRACT Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism. PMID:23572552

  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. PMID:27208255

  7. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae.

    PubMed

    Roque, Sylvain; Cerciat, Marie; Gaugué, Isabelle; Mora, Liliana; Floch, Aurélie G; de Zamaroczy, Miklos; Heurgué-Hamard, Valérie; Kervestin, Stephanie

    2015-01-01

    Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination. PMID:25411355

  8. Poly-A binding protein-1 localization to a subset of TDP-43 inclusions in amyotrophic lateral sclerosis occurs more frequently in patients harboring an expansion in C9orf72.

    PubMed

    McGurk, Leeanne; Lee, Virginia M; Trojanowksi, John Q; Van Deerlin, Vivianna M; Lee, Edward B; Bonini, Nancy M

    2014-09-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease in which the loss of spinal cord motor neurons leads to paralysis and death within a few years of clinical disease onset. In almost all cases of ALS, transactive response DNA binding protein of 43 kDa (TDP-43) forms cytoplasmic neuronal inclusions. A second causative gene for a subset of ALS is fused in sarcoma, an RNA binding protein that also forms cytoplasmic inclusions in spinal cord motor neurons. Poly-A binding protein-1 (PABP-1) is a marker of stress granules (i.e. accumulations of proteins and RNA indicative of translational arrest in cells under stress). We report on the colocalization of PABP-1 to both TDP-43 and fused-in-sarcoma inclusions in 4 patient cohorts: ALS without a mutation, ALS with an intermediate polyglutamine repeat expansion in ATXN2, ALS with a GGGGCC hexanucleotide repeat expansion in C9orf72, and ALS with basophilic inclusion body disease. Notably, PABP-1 colocalization to TDP-43 was twice as frequent in ALS with C9orf72 expansions compared to ALS with no mutation. This study highlights PABP-1 as a protein that is important to the pathology of ALS and indicates that the proteomic profile of TDP-43 inclusions in ALS may differ depending on the causative genetic mutation. PMID:25111021

  9. Poly(A) tail-mediated gene regulation by opposing roles of Nab2 and Pab2 nuclear poly(A)-binding proteins in pre-mRNA decay.

    PubMed

    Grenier St-Sauveur, Valérie; Soucek, Sharon; Corbett, Anita H; Bachand, François

    2013-12-01

    The 3' end of most eukaryotic transcripts is decorated by poly(A)-binding proteins (PABPs), which influence the fate of mRNAs throughout gene expression. However, despite the fact that multiple PABPs coexist in the nuclei of most eukaryotes, how functional interplay between these nuclear PABPs controls gene expression remains unclear. By characterizing the ortholog of the Nab2/ZC3H14 zinc finger PABP in Schizosaccharomyces pombe, we show here that the two major fission yeast nuclear PABPs, Pab2 and Nab2, have opposing roles in posttranscriptional gene regulation. Notably, we find that Nab2 functions in gene-specific regulation in a manner opposite to that of Pab2. By studying the ribosomal-protein-coding gene rpl30-2, which is negatively regulated by Pab2 via a nuclear pre-mRNA decay pathway that depends on the nuclear exosome subunit Rrp6, we show that Nab2 promotes rpl30-2 expression by acting at the level of the unspliced pre-mRNA. Our data support a model in which Nab2 impedes Pab2/Rrp6-mediated decay by competing with Pab2 for polyadenylated transcripts in the nucleus. The opposing roles of Pab2 and Nab2 reveal that interplay between nuclear PABPs can influence gene regulation. PMID:24081329

  10. Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

    PubMed

    Xiao, Yihong; Wu, Weining; Gao, Jiming; Smith, Nikki; Burkard, Christine; Xia, Dong; Zhang, Minxia; Wang, Chengbao; Archibald, Alan; Digard, Paul; Zhou, En-Min; Hiscox, Julian A

    2016-05-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins. PMID:26709850

  11. The Inhibition of Heat Shock Protein 90 Facilitates the Degradation of Poly-Alanine Expanded Poly (A) Binding Protein Nuclear 1 via the Carboxyl Terminus of Heat Shock Protein 70-Interacting Protein

    PubMed Central

    Shi, Chao; Huang, Xuan; Zhang, Bin; Zhu, Dan; Luo, Huqiao; Lu, Quqin; Xiong, Wen-Cheng; Mei, Lin; Luo, Shiwen

    2015-01-01

    Background Since the identification of poly-alanine expanded poly(A) binding protein nuclear 1 (PABPN1) as the genetic cause of oculopharyngeal muscular dystrophy (OPMD), considerable progress has been made in our understanding of the pathogenesis of the disease. However, the molecular mechanisms that regulate the onset and progression of the disease remain unclear. Results In this study, we show that PABPN1 interacts with and is stabilized by heat shock protein 90 (HSP90). Treatment with the HSP90 inhibitor 17-AAG disrupted the interaction of mutant PABPN1 with HSP90 and reduced the formation of intranuclear inclusions (INIs). Furthermore, mutant PABPN1 was preferentially degraded in the presence of 17-AAG compared with wild-type PABPN1 in vitro and in vivo. The effect of 17-AAG was mediated through an increase in the interaction of PABPN1 with the carboxyl terminus of heat shock protein 70-interacting protein (CHIP). The overexpression of CHIP suppressed the aggregation of mutant PABPN1 in transfected cells. Conclusions Our results demonstrate that the HSP90 molecular chaperone system plays a crucial role in the selective elimination of abnormal PABPN1 proteins and also suggest a potential therapeutic application of the HSP90 inhibitor 17-AAG for the treatment of OPMD. PMID:26414348

  12. Poly-A binding protein-1 localization to a subset of TAR DNA-binding protein of 43 kDa inclusions in amyotrophic lateral sclerosis occurs more frequently in patients harboring an expansion in C9orf72

    PubMed Central

    McGurk, Leeanne; Lee, Virginia, M.; Trojanowksi, John Q.; Van Deerlin, Vivianna M.; Lee, Edward B.; Bonini, Nancy M.

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease in which the loss of spinal cord motor neurons leads to paralysis and death within a few years of clinical disease onset. In almost all cases of ALS, TAR DNA binding protein of 43 kDa (TDP-43) forms cytoplasmic neuronal inclusions. A second causative gene for a subset of ALS is fused in sarcoma (FUS), an RNA binding protein that also forms cytoplasmic inclusions in spinal cord motor neurons. Poly A binding protein 1 (PABP-1) is a marker of stress granules, i.e. accumulations of proteins and RNA indicative of translational arrest in cells under stress. We report on the colocalization PABP-1 to both TDP-43 and FUS inclusions in 4 patient cohorts: ALS without a mutation, ALS with an intermediate poly glutamine repeat expansion in ATXN2, ALS with a GGGGCC-hexanucleotide repeat expansion in C9orf72, and ALS with basophilic inclusion body disease. Notably, PABP-1 colocalization to TDP-43 was twice as frequent in ALS with C9orf72 expansions compared to ALS with no mutation. This study highlights PABP-1 as a protein important to the pathology of ALS and indicates that the proteomic profile of TDP-43 inclusions in ALS may be different depending on the causative genetic mutation. PMID:25111021

  13. 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.

  14. Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae

    PubMed Central

    Roque, Sylvain; Cerciat, Marie; Gaugué, Isabelle; Mora, Liliana; Floch, Aurélie G.; de Zamaroczy, Miklos; Heurgué-Hamard, Valérie

    2015-01-01

    Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination. PMID:25411355

  15. Investigation of N-aryl-3-alkylidenepyrrolinones as potential Niemann-Pick type C disease therapeutics1

    PubMed Central

    Cosner, Casey C.; Markiewicz, John T.; Bourbon, Pauline; Mariani, Christopher J.; Wiest, Olaf; Rujoi, Madalina; Rosenbaum, Anton; Huang, Amy; Maxfield, Frederick R.; Helquist, Paul

    2009-01-01

    A five-step synthesis of an array of N-aryl-3-alkylidenepyrrolinones, which are potential Niemann-Pick type C (NPC) disease therapeutics, is described. The synthetic route allows for the production of analogues, including photoaffinity and biotinylated derivatives. Compound 1a increased esterification by acyl-coenzyme A:cholesteryl acyltransferase in NPC1 mutant cells. It also decreased LDL uptake and increased cholesterol efflux in both NPC1-deficient and normal cells. PMID:19772346

  16. Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

    PubMed Central

    2011-01-01

    Background Striatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between

  17. Recruitment of PI4KIIIβ to Coxsackievirus B3 Replication Organelles Is Independent of ACBD3, GBF1, and Arf1

    PubMed Central

    Dorobantu, Cristina M.; van der Schaar, Hilde M.; Ford, Lauren A.; Strating, Jeroen R. P. M.; Ulferts, Rachel; Fang, Ying; Belov, George

    2014-01-01

    ABSTRACT Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIβ (phosphatidylinositol-4-kinase IIIβ) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIβ to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIβ through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIβ. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIβ to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIβ recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIβ via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIβ. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIβ, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIβ to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these “replication organelles” depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are

  18. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.

    2012-01-01

    The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus

  19. 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

  20. Greater Monoamine Oxidase A Binding in Alcohol Dependence

    PubMed Central

    Matthews, Brittany A.; Kish, Stephen J.; Xu, Xin; Boileau, Isabelle; Rusjan, Pablo M.; Wilson, Alan A.; DiGiacomo, Dan; Houle, Sylvain; Meyer, Jeffrey H.

    2016-01-01

    Background Alcohol dependence (AD) is a multiorgan disease in which excessive oxidative stress and apoptosis are implicated. Monoamine oxidase A (MAO-A) is an important enzyme on the outer mitochondrial membrane that participates in the cellular response to oxidative stress and mitochondrial toxicity. It is unknown whether MAO-A levels are abnormal in AD. We hypothesized that MAO-A VT, an index of MAO-A level, is elevated in the prefrontal cortex (PFC) during AD, because markers of greater oxidative stress and apoptosis are reported in the brain in AD and a microarray analysis reported greater MAO-A messenger RNA in the PFC of rodents exposed to alcohol vapor. Methods Sixteen participants with alcohol dependence and 16 healthy control subjects underwent [11C]-harmine positron emission tomography. All were nonsmoking, medication- and drug-free, and had no other past or present psychiatric or medical illnesses. Results MAO-A VT was significantly greater in the PFC (37%, independent samples t test, t30 = 3.93, p < .001), and all brain regions analyzed (mean 32%, multivariate analysis of variance, F7,24 = 3.67, p = .008). Greater duration of heavy drinking correlated positively with greater MAO-A VT in the PFC (r = .67, p = .005) and all brain regions analyzed (r = .73 to .57, p = .001–.02). Conclusions This finding represents a new pathological marker present in AD that is therapeutically targetable through direct inhibition or by novel treatments toward oxidative/pro-apoptotic processes implicated by MAO-A overexpression. PMID:24269057

  1. Poly(A) binding proteins: are they all created equal?

    PubMed

    Goss, Dixie J; Kleiman, Frida Esther

    2013-01-01

    The PABP family of proteins were originally thought of as a simple shield for the mRNA poly(A) tail. Years of research have shown that PABPs interact not only with the poly(A) tail, but also with specific sequences in the mRNA, having a general and specific role on the metabolism of different mRNAs. The complexity of PABPs function is increased by the interactions of PABPs with factors involved in different cellular functions. PABPs participate in all the metabolic pathways of the mRNA: polyadenylation/deadenylation, mRNA export, mRNA surveillance, translation, mRNA degradation, microRNA-associated regulation, and regulation of expression during development. In this review, we update information on the roles of PABPs and emerging data on the specific interactions of PABP homologs. Specific functions of individual members of PABPC family in development and viral infection are beginning to be elucidated. However, the interactions are complex and recent evidence for exchange of nuclear and cytoplasmic forms of the proteins, as well as post-translational modifications, emphasize the possibilities for fine-tuning the PABP metabolic network. PMID:23424172

  2. 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.

  3. A binding question: the evolution of the receptor concept

    PubMed Central

    Maehle, Andreas-Holger

    2009-01-01

    In present-day pharmacology and medicine, it is usually taken for granted that cells contain a host of highly specific receptors. These are defined as proteins on or within the cell that bind with specificity to particular drugs, chemical messenger substances or hormones and mediate their effects on the body. However, it is only relatively recently that the notion of drug-specific receptors has become widely accepted, with considerable doubts being expressed about their existence as late as the 1960s. When did the receptor concept emerge, how did it evolve and why did it take so long to become established? PMID:19837460

  4. 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

  5. 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

  6. Bastadins, brominated-tyrosine derivatives, suppress accumulation of cholesterol ester in macrophages.

    PubMed

    Eguchi, Keisuke; Kato, Hikaru; Fujiwara, Yukio; Losung, Fitje; Mangindaan, Remy E P; de Voogd, Nicole J; Takeya, Motohiro; Tsukamoto, Sachiko

    2015-11-15

    The formation of foam cells in macrophages has been suggested to play an essential role in the progression of early atherosclerotic lesions in vivo and, thus, its suppression is considered to be one of the major approaches for the treatment of atherosclerosis. We isolated eight brominated-tyrosine derivatives, bastadins, from the EtOH extract of the marine sponge Ianthella vasta as inhibitors of the formation of foam cells induced by acetylated low-density lipoproteins in human monocyte-derived macrophages. Bastadin 6 was the strongest inhibitor of foam cell formation due to its suppression of acyl-coenzyme A:cholesterol acyltransferase. PMID:26403929

  7. 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

  8. 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.

  9. Characterization of the Mycobacterial Acyl-CoA Carboxylase Holo Complexes Reveals Their Functional Expansion into Amino Acid Catabolism

    PubMed Central

    Ehebauer, Matthias T.; Zimmermann, Michael; Jakobi, Arjen J.; Noens, Elke E.; Laubitz, Daniel; Cichocki, Bogdan; Marrakchi, Hedia; Lanéelle, Marie-Antoinette; Daffé, Mamadou; Sachse, Carsten; Dziembowski, Andrzej; Sauer, Uwe; Wilmanns, Matthias

    2015-01-01

    Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these - the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α−β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications. PMID:25695631

  10. An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

    PubMed Central

    Daniel, Jaiyanth; Sirakova, Tatiana; Kolattukudy, Pappachan

    2014-01-01

    Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids. PMID:25490545

  11. Patatin-Related Phospholipase pPLAIIIδ Increases Seed Oil Content with Long-Chain Fatty Acids in Arabidopsis1[C][W][OA

    PubMed Central

    Li, Maoyin; Bahn, Sung Chul; Fan, Chuchuan; Li, Jia; Phan, Tien; Ortiz, Michael; Roth, Mary R.; Welti, Ruth; Jaworski, Jan; Wang, Xuemin

    2013-01-01

    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. PMID:23542150

  12. Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

    PubMed Central

    Inaba, Tomohiro; Tokumoto, Yuta; Miyazaki, Yusuke; Inoue, Naoyuki; Maseda, Hideaki; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo

    2013-01-01

    Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species. PMID:24038682

  13. Penicillium griseofulvum F1959, high-production strain of pyripyropene a, specific inhibitor of acyl-CoA: cholesterol acyltransferase 2.

    PubMed

    Choi, Jung Ho; Rho, Mun-Chual; Lee, Seung Woong; Choi, Ji Na; Lee, Hee Jeong; Bae, Kyung Sook; Kim, Koanhoi; Kim, Young Kook

    2008-10-01

    Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification and plays an important role in the intestinal absorption of cholesterol, hepatic production of lipoproteins, and accumulation of cholesteryl ester within cells. During the course of screening to find ACAT inhibitors from microbial sources, the present authors isolated pyripyropene A from Penicillium griseofulvum F1959. Pyripyropene A, an ACAT2-specific inhibitor, has already been produced from Aspergillus fumigatus. Yet, Aspergillus fumigatus is a pathogen and only produces a limited amount of pyripyropene A, making the isolation of pyripyropene A troublesome. In contrast, Penicillium griseofulvum F1959 was found to produce approximately 28 times more pyripyropene A than Aspergillus fumigatus, plus this report also describes the ideal conditions for the production of pyripyropene A by Penicillium griseofulvum F1959 and its subsequent purification. PMID:18955816

  14. Assignment of function to Histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group.†

    PubMed Central

    Shim, Da Jeong; Nemeria, Natalia S.; Balakrishnan, Anand; Patel, Hetalben; Song, Jaeyoung; Wang, Junjie; Jordan, Frank; Farinas, Edgardo T.

    2011-01-01

    The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced by hydrophobic residues of similar molecular volume. To interrogate whether the second component would enable synthesis of acyl-coenzymeA derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the ‘gatekeeper’ for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate, but E2o for 2-oxoglutarate. PMID:21809826

  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. Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation

    PubMed Central

    Mera, Paola E.; Kalogeraki, Virginia S.; Shapiro, Lucy

    2014-01-01

    During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation. PMID:25349407

  17. Hand proximity differentially affects visual working memory for color and orientation in a binding task.

    PubMed

    Kelly, Shane P; Brockmole, James R

    2014-01-01

    Observers determined whether two sequentially presented arrays of six lines were the same or different. Differences, when present, involved either a swap in the color of two lines or a swap in the orientation of two lines. Thus, accurate change detection required the binding of color and orientation information for each line within visual working memory. Holding viewing distance constant, the proximity of the arrays to the hands was manipulated. Placing the hands near the to-be-remembered array decreased participants' ability to remember color information, but increased their ability to remember orientation information. This pair of results indicates that hand proximity differentially affects the processing of various types of visual information, a conclusion broadly consistent with functional and anatomical differences in the magnocellular and parvocellular pathways. It further indicates that hand proximity affects the likelihood that various object features will be encoded into integrated object files. PMID:24795671

  18. Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome

    PubMed Central

    Waldminghaus, Torsten; Weigel, Christoph; Skarstad, Kirsten

    2012-01-01

    In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork. We show that this is, indeed, the case by using global ChIP on Chip analysis of SeqA in cells synchronized regarding DNA replication. To assess hemimethylation, we developed the first genome-wide method for methylation analysis in bacteria. Since loss of the SeqA protein affects growth rate only during rapid growth when cells contain multiple replication forks, a comparison of rapid and slow growth was performed. In cells with six replication forks per chromosome, the two old forks were found to bind surprisingly little SeqA protein. Cell cycle analysis showed that loss of SeqA from the old forks did not occur at initiation of the new forks, but instead occurs at a time point coinciding with the end of SeqA-dependent origin sequestration. The finding suggests simultaneous origin de-sequestration and loss of SeqA from old replication forks. PMID:22373925

  19. Dopamine receptors in the guinea-pig heart. A binding study

    SciTech Connect

    Sandrini, M.; Benelli, A.; Baraldi, M.

    1984-10-29

    The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using /sup 3/H-dopamine and /sup 3/H-spiperone as radioligand. /sup 3/H-Dopamine bound specifically to heart membranes while /sup 3/H-spiperone did not. A Scatchard analysis of /sup 3/H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (K/sub d/ = 1.2 nM, B/sub mx/ = 52.9 fmol/mg prot.) and low- (K/sub d/ = 11.8 nM, B/sub mx/ = 267.3 fmol/gm prot.) affinity binding sites, respectively. The charactization of the high-affinity component of /sup 3/H-dopamine binding indicated tha

  20. 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

  1. Identification of a Binding Site for Unsaturated Fatty Acids in the Orphan Nuclear Receptor Nurr1.

    PubMed

    de Vera, Ian Mitchelle S; Giri, Pankaj K; Munoz-Tello, Paola; Brust, Richard; Fuhrmann, Jakob; Matta-Camacho, Edna; Shang, Jinsai; Campbell, Sean; Wilson, Henry D; Granados, Juan; Gardner, William J; Creamer, Trevor P; Solt, Laura A; Kojetin, Douglas J

    2016-07-15

    Nurr1/NR4A2 is an orphan nuclear receptor, and currently there are no known natural ligands that bind Nurr1. A recent metabolomics study identified unsaturated fatty acids, including arachidonic acid and docosahexaenoic acid (DHA), that interact with the ligand-binding domain (LBD) of a related orphan receptor, Nur77/NR4A1. However, the binding location and whether these ligands bind other NR4A receptors were not defined. Here, we show that unsaturated fatty acids also interact with the Nurr1 LBD, and solution NMR spectroscopy reveals the binding epitope of DHA at its putative ligand-binding pocket. Biochemical assays reveal that DHA-bound Nurr1 interacts with high affinity with a peptide derived from PIASγ, a protein that interacts with Nurr1 in cellular extracts, and DHA also affects cellular Nurr1 transactivation. This work is the first structural report of a natural ligand binding to a canonical NR4A ligand-binding pocket and indicates a natural ligand can bind and affect Nurr1 function. PMID:27128111

  2. Sensitive detection of Bacillus anthracis using a binding protein originating from gamma-phage.

    PubMed

    Fujinami, Yoshihito; Hirai, Yoshikazu; Sakai, Ikuko; Yoshino, Mineo; Yasuda, Jiro

    2007-01-01

    Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C-terminal region of gamma-phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156-233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the gamma-phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the gamma-phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis. PMID:17310083

  3. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... make only a partial delivery of the shipment, you may not demand upon delivery full payment of the... with the following 11 requirements: (1) You must base the binding estimate on the physical survey... clearly indicate on its face that the charges shown apply only to those services specifically...

  4. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... shipment, you may not demand upon delivery full payment of the binding estimate. You may demand only a... with the following 11 requirements: (1) You must base the binding estimate on the physical survey... clearly indicate on its face that the charges shown apply only to those services specifically...

  5. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... make only a partial delivery of the shipment, you may not demand upon delivery full payment of the... with the following 11 requirements: (1) You must base the binding estimate on the physical survey... clearly indicate on its face that the charges shown apply only to those services specifically...

  6. 49 CFR 375.403 - How must I provide a binding estimate?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... make only a partial delivery of the shipment, you may not demand upon delivery full payment of the... with the following 11 requirements: (1) You must base the binding estimate on the physical survey... clearly indicate on its face that the charges shown apply only to those services specifically...

  7. Fusobacterium nucleatum adhesin FadA binds vascular-endothelial cadherin and alters endothelial integrity

    PubMed Central

    Fardini, Yann; Wang, Xiaowei; Témoin, Stéphanie; Nithianantham, Stanley; Lee, David; Shoham, Menachem; Han, Yiping W.

    2011-01-01

    SUMMARY Fusobacterium nucleatum is a gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA co-localized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and E. coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an “enabler” for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens. PMID:22040113

  8. A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor

    PubMed Central

    VAN GALEN, PHILIP J. M.; VAN BERGEN, ANDREW H.; GALLO-RODRIGUEZ, CAROLA; MELMAN, NELI; OLAH, MARK E.; IJZERMAN, AD P.; STILES, GARY L.; JACOBSON, KENNETH A.

    2012-01-01

    SUMMARY A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands. PMID:8022403

  9. 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

  10. Being a binding site: characterizing residue composition of binding sites on proteins.

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-01-01

    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. PMID:18305831

  11. Apolipoprotein E includes a binding site which is recognized by several amyloidogenic polypeptides.

    PubMed Central

    Baumann, M H; Kallijärvi, J; Lankinen, H; Soto, C; Haltia, M

    2000-01-01

    Inheritance of the apolipoprotein E (apoE) epsilon 4 allele is a risk factor for late-onset Alzheimer's disease (AD). Biochemically apoE is present in AD plaques and neurofibrillary tangles of the AD brain. There is a high avidity and specific binding of apoE and the amyloid beta-peptide (A beta). In addition to AD apoE is also present in many other cerebral and systemic amyloidoses, Down's syndrome and prion diseases but the pathophysiological basis for its presence is still unknown. In the present study we have compared the interaction of apoE with A beta, the gelsolin-derived amyloid fragment AGel(183-210) and the amyloidogenic prion fragments PrP(109-122) and PrP(109-141). We show that, similar to A beta, also AGel and PrP fragments can form a complex with apoE, and that the interaction between apoE and the amyloidogenic protein fragments is mediated through the same binding site on apoE. We also show that apoE increases the thioflavin-T fluorescence of PrP and AGel and that apoE influences the content of beta-sheet conformation of these amyloidogenic fragments. Our results indicate that amyloids and amyloidogenic prion fragments share a similar structural motif, which is recognized by apoE, possibly through a single binding site, and that this motif is also responsible for the amyloidogenicity of these fragments. PMID:10861213

  12. 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.

  13. Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds.

    PubMed

    Davidenko, Natalia; Bax, Daniel V; Schuster, Carlos F; Farndale, Richard W; Hamaia, Samir W; Best, Serena M; Cameron, Ruth E

    2016-01-01

    Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 μm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality. PMID:26676860

  14. Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.

    PubMed

    Sakaguchi, K; Zambrano, N; Baldwin, E T; Shapiro, B A; Erickson, J W; Omichinski, J G; Clore, G M; Gronenborn, A M; Appella, E

    1993-06-01

    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. PMID:8506369

  15. 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

  16. 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. PMID:22568044

  17. Human IgA-binding Peptides Selected from Random Peptide Libraries

    PubMed Central

    Hatanaka, Takaaki; Ohzono, Shinji; Park, Mirae; Sakamoto, Kotaro; Tsukamoto, Shogo; Sugita, Ryohei; Ishitobi, Hiroyuki; Mori, Toshiyuki; Ito, Osamu; Sorajo, Koichi; Sugimura, Kazuhisa; Ham, Sihyun; Ito, Yuji

    2012-01-01

    Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1–A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (Kd = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (Kd = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination. PMID:23076147

  18. 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

  19. The CENP-T/-W complex is a binding partner of the histone chaperone FACT.

    PubMed

    Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J; Sullivan, Kevin F; Almouzni, Geneviève

    2016-06-01

    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

  20. 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.

  1. Lipoprotein(a) binds to fibronectin and has serine proteinase activity capable of cleaving it.

    PubMed Central

    Salonen, E M; Jauhiainen, M; Zardi, L; Vaheri, A; Ehnholm, C

    1989-01-01

    The plasma concentration of human lipoprotein(a) [Lp(a)] is correlated with the risk of heart disease. A distinct feature of the Lp(a) particle is the apolipoprotein (a) [apo(a)], which is associated with apoB-100, the main protein component of low-density lipoprotein. We now report that apo(a), which has extensive homology to plasminogen, binds to immobilized fibronectin. The binding of Lp(a) was localized to the C-terminal heparin-binding domain of fibronectin. Incubation of Lp(a) with fibronectin resulted in fragmentation of fibronectin. The cleavage pattern, as visualized by gel electrophoresis and immunoblotting, was reproducibly obtained with Lp(a) purified from five different individuals and was distinct from that obtained upon proteolysis of fibronectin by plasmin or kallikrein. The use of synthetic peptide substrates demonstrated that the amino acid specificity for Lp(a) was arginine rather than lysine. The proteolytic activity of Lp(a) was localized to apo(a) and experiments with inhibitors indicated that the proteolytic activity was of serine proteinase-type. Images PMID:2531657

  2. 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.

  3. 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

    1994-07-01

    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. PMID:8023141

  4. Identification of C1q as a Binding Protein for Advanced Glycation End Products.

    PubMed

    Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

    2016-01-26

    Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway. PMID:26731343

  5. 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. PMID:20181873

  6. 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

  7. Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

    PubMed

    Jasieniecka-Gazarkiewicz, Katarzyna; Demski, Kamil; Lager, Ida; Stymne, Sten; Banaś, Antoni

    2016-01-01

    Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme. PMID:26643989

  8. 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. PMID:25659146

  9. Gene Expression Patterns Associated with Peroxisome Proliferator-activated Receptor (PPAR) Signaling in the Longissimus dorsi of Hanwoo (Korean Cattle)

    PubMed Central

    Lim, Dajeong; Chai, Han-Ha; Lee, Seung-Hwan; Cho, Yong-Min; Choi, Jung-Woo; Kim, Nam-Kuk

    2015-01-01

    Adipose tissue deposited within muscle fibers, known as intramuscular fat (IMF or marbling), is a major determinant of meat quality and thereby affects its economic value. The biological mechanisms that determine IMF content are therefore of interest. In this study, 48 genes involved in the bovine peroxisome proliferator-activated receptor signaling pathway, which is involved in lipid metabolism, were investigated to identify candidate genes associated with IMF in the longissimus dorsi of Hanwoo (Korean cattle). Ten genes, retinoid X receptor alpha, peroxisome proliferator-activated receptor gamma (PPARG), phospholipid transfer protein, stearoyl-CoA desaturase, nuclear receptor subfamily 1 group H member 3, fatty acid binding protein 3 (FABP3), carnitine palmitoyltransferase II, acyl-Coenzyme A dehydrogenase long chain (ACADL), acyl-Coenzyme A oxidase 2 branched chain, and fatty acid binding protein 4, showed significant effects with regard to IMF and were differentially expressed between the low- and high-marbled groups (p<0.05). Analysis of the gene co-expression network based on Pearson’s correlation coefficients identified 10 up-regulated genes in the high-marbled group that formed a major cluster. Among these genes, the PPARG-FABP4 gene pair exhibited the strongest correlation in the network. Glycerol kinase was found to play a role in mediating activation of the differentially expressed genes. We categorized the 10 significantly differentially expressed genes into the corresponding downstream pathways and investigated the direct interactive relationships among these genes. We suggest that fatty acid oxidation is the major downstream pathway affecting IMF content. The PPARG/RXRA complex triggers activation of target genes involved in fatty acid oxidation resulting in increased triglyceride formation by ATP production. Our findings highlight candidate genes associated with the IMF content of the loin muscle of Korean cattle and provide insight into the

  10. Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by Microarray-Bioinformatics analysis.

    PubMed

    Yeh, Ching-Sheng; Wang, Jaw-Yuan; Cheng, Tian-Lu; Juan, Chin-Hung; Wu, Chan-Han; Lin, Shiu-Ru

    2006-02-28

    The present study systematically explored metabolic pathways and altered expressions of genes speculatively participating in colorectal carcinogenesis by using a Microarray-Bioinformatic analysis methods. The results revealed that 157 genes were up-regulated and 281 genes were down-regulated in colorectal cancer (CRC). Gene Ontology (GO) and relevant bioinformatics tools indicated that the functional category to which 438 genes (12%; 438/3800) of the most frequent alteration belonged was metabolism. The analysis of 10 colorectal cancer tissue specimens demonstrated that genes involved in fatty acid metabolic pathways had high rates of overexpression. In addition, we stimulated CRL-1790 cell line with linoleic acid (a polyunsaturated fatty acid) for 12, 24, 48 and 72 h. Cell proliferation was elevated by 5, 25, 28 and 31% (P<0.05), respectively. Further analyses revealed that the genes increasingly expressed in the cell line included enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase (EHHADH), enoyl Coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1); glutaryl-Coenzyme A dehydrogenase (GCDH), acyl-Coenzyme A oxidase 2, branched chain (ACOX2); acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor (ACADS); carnitine palmitoyltransferase 1B (CPT1B), acyl-CoA synthetase long-chain family member 5 (ACSL5), and cytochrome P450, family 4, subfamily A, and polypeptide 11 (CYP4A11) genes. This indicated that the stimulating effect of linoleic acid on cell proliferation was due to interference with the metabolic pathway of fatty acid metabolism. In conclusion, genes with altered expression levels in CRC were mainly associated with fatty acid metabolic pathways speculated to have an important role linked to carcinogenesis. PMID:15885896

  11. Green tea extract suppresses adiposity and affects the expression of lipid metabolism genes in diet-induced obese zebrafish

    PubMed Central

    2012-01-01

    Background Visceral fat accumulation is one of the most important predictors of mortality in obese populations. Administration of green tea extract (GTE) can reduce body fat and reduce the risk of obesity-related diseases in mammals. In this study, we investigated the effects and mechanisms of GTE on adiposity in diet-induced obese (DIO) zebrafish. Methods Zebrafish at 3.5 to 4.5 months post-fertilization were allocated to four groups: non-DIO, DIO, DIO + 0.0025%GTE, and DIO + 0.0050%GTE. The non-DIO group was fed freshly hatched Artemia once daily (5 mg cysts/fish daily) for 40 days. Zebrafish in the three DIO groups were fed freshly hatched Artemia three times daily (60 mg cysts/fish daily). Zebrafish in the DIO + 0.0025%GTE and DIO + 0.0050%GTE groups were exposed to GTE after the start of feeding three times daily for 40 days. Results Three-dimensional microcomputed tomography analysis showed that GTE exposure significantly decreased the volume of visceral but not subcutaneous fat tissue in DIO zebrafish. GTE exposure increased hepatic expression of the lipid catabolism genes ACOX1 (acyl-coenzyme A oxidase 1, palmitoyl), ACADM (acyl-coenzyme A dehydrogenase, c-4 to c-12 straight chain), and PPARA (peroxisome proliferator-activated receptor alpha). GTE exposure also significantly decreased the visceral fat expression of SOCS3 (suppressor of cytokine signaling 3b) which inhibits leptin signaling. Conclusions The present results are consistent with those seen in mammals treated with GTE, supporting the validity of studying the effects of GTE in DIO zebrafish. Our results suggest that GTE exerts beneficial effects on adiposity, possibly by altering the expression of lipid catabolism genes and SOCS3. PMID:22871059

  12. A binding site outside the canonical PDZ domain determines the specific interaction between Shank and SAPAP and their function.

    PubMed

    Zeng, Menglong; Shang, Yuan; Guo, Tingfeng; He, Qinghai; Yung, Wing-Ho; Liu, Kai; Zhang, Mingjie

    2016-05-31

    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

  13. Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal-specific tubulin.

    PubMed

    Avdoshina, Valeria; Taraballi, Francesca; Dedoni, Simona; Corbo, Claudia; Paige, Mikell; Saygideğer Kont, Yasemin; Üren, Aykut; Tasciotti, Ennio; Mocchetti, Italo

    2016-04-01

    Human immunodeficiency virus-1 (HIV) promotes synaptic simplification and neuronal apoptosis, and causes neurological impairments termed HIV-associated neurological disorders. 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 HIV-associated neurocognitive disorders subjects. This 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 cross-linked 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. We propose a novel direct mechanism of human immunodeficiency virus neurotoxicity. Our data show that the viral protein gp120 binds to neuronal specific tubulin β-3 and blocks microtubule transport. Displacing gp120 from binding to tubulin by a small peptide prevents gp120-mediated neuronal loss. Our study reveals a novel target for developing adjunct therapies against viral infection that promotes neurocognitive disorders. PMID:26826352

  14. (1) Request for a binding decision on whether Ferdinanda (Compositae) and Ferdinandea (Rubiaceae) are sufficiently alike to be confused

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  15. A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro

    PubMed Central

    Raiber, Eun-Ang; Kranaster, Ramon; Lam, Enid; Nikan, Mehran; Balasubramanian, Shankar

    2012-01-01

    SP1 is a ubiquitous transcription factor that is involved in the regulation of various house-keeping genes. It is known that it acts by binding to a double-stranded consensus motif. Here, we have discovered that SP1 binds also to a non-canonical DNA structure, a G-quadruplex, with high affinity. In particular, we have studied the SP1 binding site within the promoter region of the c-KIT oncogene and found that this site can fold into an anti-parallel two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts, together with biophysical binding assays revealed that SP1 has a comparable binding affinity for this G-quadruplex structure and the canonical SP1 duplex sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1 binding sites overlap with G-quadruplex forming sequences. Furthermore, while many of these immuoprecipitated sequences (36%) even lack the minimal SP1 consensus motif, 5′-GGGCGG-3′, we have shown that 77% of them are putative G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both, canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we hypothesize that both types of interactions may occur in cells. PMID:22021377

  16. 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. PMID:25709405

  17. A binding mode hypothesis of tiagabine confirms liothyronine effect on γ-aminobutyric acid transporter 1 (GAT1).

    PubMed

    Jurik, Andreas; Zdrazil, Barbara; Holy, Marion; Stockner, Thomas; Sitte, Harald H; Ecker, Gerhard F

    2015-03-12

    Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand-transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure-activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone. PMID:25679268

  18. At the right place at the right time: novel CENP-A binding proteins shed light on centromere assembly.

    PubMed

    Silva, Mariana C C; Jansen, Lars E T

    2009-10-01

    Centromeres, the chromosomal loci that form the sites of attachment for spindle microtubules during mitosis, are identified by a unique chromatin structure generated by nucleosomes containing the histone H3 variant CENP-A. The apparent epigenetic mode of centromere inheritance across mitotic and meiotic divisions has generated much interest in how CENP-A assembly occurs and how structurally divergent centromeric nucleosomes can specify the centromere complex. Although a substantial number of proteins have been implicated in centromere assembly, factors that can bind CENP-A specifically and deliver nascent protein to the centromere were, thus far, lacking. Several recent reports on experiments in fission yeast and human cells have now shown significant progress on this problem. Here, we discuss these new developments and their implications for epigenetic centromere inheritance. PMID:19590885

  19. Cruentaren A Binds F1F0 ATP Synthase To Modulate the Hsp90 Protein Folding Machinery

    PubMed Central

    2015-01-01

    The molecular chaperone Hsp90 requires the assistance of immunophilins, co-chaperones, and partner proteins for the conformational maturation of client proteins. Hsp90 inhibition represents a promising anticancer strategy due to the dependence of numerous oncogenic signaling pathways upon Hsp90 function. Historically, small molecules have been designed to inhibit ATPase activity at the Hsp90 N-terminus; however, these molecules also induce the pro-survival heat shock response (HSR). Therefore, inhibitors that exhibit alternative mechanisms of action that do not elicit the HSR are actively sought. Small molecules that disrupt Hsp90-co-chaperone interactions can destabilize the Hsp90 complex without induction of the HSR, which leads to inhibition of cell proliferation. In this article, selective inhibition of F1F0 ATP synthase by cruentaren A was shown to disrupt the Hsp90-F1F0 ATP synthase interaction and result in client protein degradation without induction of the HSR. PMID:24450340

  20. Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition.

    PubMed

    Parker, Matthew W; Xu, Ping; Guo, Hou-Fu; Vander Kooi, Craig W

    2012-01-01

    Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3. PMID:23145112

  1. Structural basis for selective vascular endothelial growth factor-A (VEGF-A) binding to neuropilin-1.

    PubMed

    Parker, Matthew W; Xu, Ping; Li, Xiaobo; Vander Kooi, Craig W

    2012-03-30

    Neuropilin-1 (Nrp1) is an essential receptor for angiogenesis that binds to VEGF-A. Nrp1 binds directly to VEGF-A with high affinity, but the nature of their selective binding has remained unclear. Nrp1 was initially reported to bind to the exon 7-encoded region of VEGF-A and function as an isoform-specific receptor for VEGF-A(164/165). Recent data have implicated exon 8-encoded residues, which are found in all proangiogenic VEGF-A isoforms, in Nrp binding. We have determined the crystal structure of the exon 7/8-encoded VEGF-A heparin binding domain in complex with the Nrp1-b1 domain. This structure clearly demonstrates that residues from both exons 7 and 8 physically contribute to Nrp1 binding. Using an in vitro binding assay, we have determined the relative contributions of exon 7- and 8-encoded residues. We demonstrate that the exon 8-encoded C-terminal arginine is essential for the interaction of VEGF-A with Nrp1 and mediates high affinity Nrp binding. Exon 7-encoded electronegative residues make additional interactions with the L1 loop of Nrp1. Although otherwise conserved, the primary sequences of Nrp1 and Nrp2 differ significantly in this region. We further show that VEGF-A(164) binds 50-fold more strongly to Nrp1 than Nrp2. Direct repulsion between the electronegative exon 7-encoded residues of the heparin binding domain and the electronegative L1 loop found only in Nrp2 is found to significantly contribute to the observed selectivity. The results reveal the basis for the potent and selective binding of VEGF-A(164) to Nrp1. PMID:22318724

  2. 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

  3. Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria.

    PubMed

    Akbar, S Md; Sreeramulu, K; Sharma, Hari C

    2016-06-01

    Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery. PMID:26905428

  4. Bisphenol A binds to Ras proteins and competes with guanine nucleotide exchange: implications for GTPase-selective antagonists.

    PubMed

    Schöpel, Miriam; Jockers, Katharina F G; Düppe, Peter M; Autzen, Jasmin; Potheraveedu, Veena N; Ince, Semra; Yip, King Tuo; Heumann, Rolf; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2013-12-12

    We show for the first time that bisphenol A (10) has the capacity to interact directly with K-Ras and that Rheb weakly binds to bisphenol A (10) and 4,4'-biphenol derivatives. We have characterized these interactions at atomic resolution suggesting that these compounds sterically interfere with the Sos-mediated nucleotide exchange in H- and K-Ras. We show that 4,4'-biphenol (5) selectively inhibits Rheb signaling and induces cell death suggesting that this compound might be a novel candidate for treatment of tuberous sclerosis-mediated tumor growth. Our results propose a new mode of action for bisphenol A (10) that advocates a reduced exposure to this compound in our environment. Our data may lay the foundation for the future design of GTPase-selective antagonists with higher affinity to benefit of the treatment of cancer because K-Ras inhibition is regarded to be a promising strategy with a potential therapeutic window for targeting Sos in Ras-driven tumors. PMID:24266771

  5. Designing a binding interface for control of cancer cell adhesion via 3D topography and metabolic oligosaccharide engineering.

    PubMed

    Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J

    2011-08-01

    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, Ac(5)ManNTGc, a thiol-bearing analog 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 bio-orthogonal 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

  6. 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.

  7. 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

  8. Nuclear DNA helicase II (RNA helicase A) binds to an F-actin containing shell that surrounds the nucleolus.

    PubMed

    Zhang, Suisheng; Köhler, Carsten; Hemmerich, Peter; Grosse, Frank

    2004-02-15

    Nuclear DNA helicase II (NDH II), alternatively named RNA helicase A (RHA), is an F-actin binding protein that is particularly enriched in the nucleolus of mouse cells. Here, we show that the nucleolar localization of NDH II of murine 3T3 cells depended on an ongoing rRNA synthesis. NDH II migrated out of the nucleolus after administration of 0.05 microg/ml actinomycin D, while nucleolin and the upstream binding factor (UBF) remained there. In S phase-arrested mouse cells, NDH II was frequently found at the nucleolar periphery, where it was accompanied by newly synthesized nucleolar RNA. Human NDH II was mainly distributed through the whole nucleoplasm and not enriched in the nucleoli. However, in the human breast carcinoma cell line MCF-7, NDH II was also found at the nucleolar periphery, together with the tumor suppressor protein p53. Both NDH II and p53 were apparently attached to the F-actin-based filamentous network that surrounded the nucleoli. Accordingly, this subnuclear structure was sensitive to F-actin depolymerizing agents. Depolymerization with gelsolin led to a striking accumulation of NDH II in the nucleoli of MCF-7 cells. This effect was abolished by RNase, which extensively released nucleolus-bound NDH II when added together with gelsolin. Taken together, these results support the idea that an actin-based filamentous network may anchor NDH II at the nucleolar periphery for pre-ribosomal RNA processing, ribosome assembly, and/or transport. PMID:14729462

  9. Stimulation of Tetrabromobisphenol A Binding to Soil Humic Substances by Birnessite and the Chemical Structure of the Bound Residues.

    PubMed

    Tong, Fei; Gu, Xueyuan; Gu, Cheng; Xie, Jinyu; Xie, Xianchuan; Jiang, Bingqi; Wang, Yongfeng; Ertunc, Tanya; Schäffer, Andreas; Ji, Rong

    2016-06-21

    Studies have shown the main fate of the flame retardant tetrabromobisphenol A (TBBPA) in soils is the formation of bound residues, and mechanisms on it are less-understood. This study investigated the effect of birnessite (δ-MnO2), a naturally occurring oxidant in soils, on the formation of bound residues. (14)C-labeled TBBPA was used to investigate the pH dependency of TBBPA bound-residue formation to two soil humic acids (HAs), Elliott soil HA and Steinkreuz soil HA, in the presence of δ-MnO2. The binding of TBBPA and its transformation products to both HAs was markedly increased (3- to 17-fold) at all pH values in the presence of δ-MnO2. More bound residues were formed with the more aromatic Elliott soil HA than with Steinkreuz soil HA. Gel-permeation chromatography revealed a uniform distribution of the bound residues within Steinkreuz soil HA and a nonuniform distribution within Elliott soil HA. (13)C NMR spectroscopy of (13)C-TBBPA residues bound to (13)C-depleted HA suggested that in the presence of δ-MnO2, binding occurred via ester and ether and other types of covalent bonds besides HA sequestration. The insights gained in this study contribute to an understanding of the formation of TBBPA bound residues facilitated by δ-MnO2. PMID:27223831

  10. Greater Monoamine Oxidase A Binding in Perimenopausal Age as Measured With Carbon 11–Labeled Harmine Positron Emission Tomography

    PubMed Central

    Rekkas, Paraskevi Vivien; Wilson, Alan A.; Lee, Vivian Wai Han; Yogalingam, Priyanga; Sacher, Julia; Rusjan, Pablo; Houle, Sylvain; Stewart, Donna E.; Kolla, Nathan J.; Kish, Stephen; Chiuccariello, Lina; Meyer, Jeffrey H.

    2016-01-01

    IMPORTANCE Perimenopause is a period of high risk for mood disorders, and it has been proposed that perimenopause is also a window of risk for processes linked to later dementia. However, in human perimenopause, the neurobiological changes implicated in the genesis of mood disorders or dementia have not been identified. Monoamine oxidase A (MAO-A) is an important brain enzyme that creates oxidative stress, influences apoptosis, and metabolizes monoamines. After declines in estrogen level, MAO-A density may be elevated for a month or longer, and repeated declines in estrogen level occur with greater magnitude during perimenopause. OBJECTIVE To investigate whether MAO-A total distribution volume (VT), an index of MAO-A density, is elevated in women of perimenopausal age (41–51 years). DESIGN, SETTING, AND PARTICIPANTS In a cross-sectional study at a tertiary care psychiatric hospital, 58 women underwent carbon 11–labeled harmine positron emission tomography. These included 19 young women of reproductive age (mean [SD], 28.26 [5.05] years), 27 women of perimenopausal age (mean [SD] age, 45.21 [3.41] years; including 14 women with change in menstrual cycle length with a mean [SD] age of 45.50 [4.00] years and 13 women with no change in menstrual cycle length with a mean [SD] age of 44.92 [2.81] years), and 12 women in menopause (mean [SD] age, 56.25 [3.19] years). MAIN OUTCOMES AND MEASURES Values of MAO-A VT in the prefrontal cortex, anterior cingulate cortex, dorsal striatum, ventral striatum, thalamus, hippocampus, and midbrain. RESULTS On average, MAO-A VT in perimenopausal age was elevated by 34% compared with reproductive age and by 16% compared with menopause (multivariate analysis of variance, group effect, F16,94 = 3.03; P < .001). Within the perimenopausal age group, meeting Stages of Reproductive Aging Workshop criteria for perimenopause, which is mainly based on menstrual cycle length, was not associated with MAO-A VT (F8,18 = 0.548; P = .81) but tendency to cry was positively correlated with MAO-A VT in the prefrontal cortex (r = 0.54; P = .008). CONCLUSIONS AND RELEVANCE To our knowledge, this is the first report of a change in a central biomarker during perimenopausal age that is also present during major depressive episodes and high-risk states for major depressive episodes. The functions of MAO-A influence oxidative stress and apoptosis, 2 processes implicated as excessive in both mood disorders and dementia. Hence, greater MAO-A VT during perimenopause may represent a new target for assessing novel interventions to prevent mood disorders and reduce longer-term risk of neurodegenerative disease. PMID:24898155

  11. 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.

  12. A Binding Mode Hypothesis of Tiagabine Confirms Liothyronine Effect on γ-Aminobutyric Acid Transporter 1 (GAT1)

    PubMed Central

    2015-01-01

    Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand–transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure–activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone. PMID:25679268

  13. 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

  14. Improved version of a binding energy formula for heavy and superheavy nuclei with Z{>=}90 and N{>=}140

    SciTech Connect

    Dong Tiekuang; Ren Zhongzhou

    2008-06-15

    A local formula of binding energy for heavy and superheavy nuclei has very recently been proposed [Dong and Ren, Phys. Rev. C 72, 064331 (2005)]. In this paper, the limit of the predictive ability of this local formula is investigated. It is found that the neutron-proton correlations should be considered when higher precision is required. On the one hand, we introduce a new term |N-Z-50|/A, and on the other hand we consider the different strengths of proton-proton, neutron-neutron, and neutron-proton pairing correlations. For the first time, the standard deviation {radical}({sigma}{sup 2}) of the binding energies for 117 nuclei with Z{>=}90 and N{>=}140 is reduced to 0.105 MeV. The {alpha} decay energies Q{sub {alpha}} and half-lives T{sub {alpha}} of nuclei with Z=102-118 are reproduced quite well. The proton drip line of superheavy elements from Md (Z=101) to Ds (Z=110) are predicted.

  15. Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

    PubMed Central

    Pintor, J.; Torres, M.; Castro, E.; Miras-Portugal, M. T.

    1991-01-01

    1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site. PMID:1912985

  16. A Binding Site on IL-17A for Inhibitory Macrocycles Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry.

    PubMed

    Espada, Alfonso; Broughton, Howard; Jones, Spencer; Chalmers, Michael J; Dodge, Jeffrey A

    2016-03-10

    Computational assessment of the IL-17A structure identified two distinct binding pockets, the β-hairpin pocket and the α-helix pocket. The β-hairpin pocket was hypothesized to be the site of binding for peptide macrocycles. Support for this hypothesis was obtained using HDX-MS which revealed protection to exchange only within the β-hairpin pocket. This data represents the first direct structural evidence of a small molecule binding site on IL-17A that functions to disrupt the interaction with its receptor. PMID:26854023

  17. A binding site for Bacillus thuringiensis Cry1Ab toxin is lost during larval development in two forest pests.

    PubMed

    Rausell, C; Martínez-Ramírez, A C; García-Robles, I; Real, M D

    2000-04-01

    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/microl, 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 (125)I-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

  18. 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

  19. The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

    PubMed

    Malarkey, Christopher S; Lionetti, Claudia; Deceglie, Stefania; Roberti, Marina; Churchill, Mair E A; Cantatore, Palmiro; Loguercio Polosa, Paola

    2016-07-01

    Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM. PMID:27101895

  20. 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. PMID:26413653

  1. 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. PMID:24559933

  2. Structural basis for the selective vascular endothelial growth factor-A (VEGF-A) binding to neuropilin-1

    SciTech Connect

    Parker, Matthew W.; Xu, Ping; Li, Xiaobo; Vander Kooi, Craig W.

    2012-07-25

    Neuropilin-1 (Nrp1) is an essential receptor for angiogenesis that binds to VEGF-A. Nrp1 binds directly to VEGF-A with high affinity, but the nature of their selective binding has remained unclear. Nrp1 was initially reported to bind to the exon 7-encoded region of VEGF-A and function as an isoform-specific receptor for VEGF-A164/165. Recent data have implicated exon 8-encoded residues, which are found in all proangiogenic VEGF-A isoforms, in Nrp binding. We have determined the crystal structure of the exon 7/8-encoded VEGF-A heparin binding domain in complex with the Nrp1-b1 domain. This structure clearly demonstrates that residues from both exons 7 and 8 physically contribute to Nrp1 binding. Using an in vitro binding assay, we have determined the relative contributions of exon 7- and 8-encoded residues. We demonstrate that the exon 8-encoded C-terminal arginine is essential for the interaction of VEGF-A with Nrp1 and mediates high affinity Nrp binding. Exon 7-encoded electronegative residues make additional interactions with the L1 loop of Nrp1. Although otherwise conserved, the primary sequences of Nrp1 and Nrp2 differ significantly in this region. We further show that VEGF-A{sub 164} binds 50-fold more strongly to Nrp1 than Nrp2. Direct repulsion between the electronegative exon 7-encoded residues of the heparin binding domain and the electronegative L1 loop found only in Nrp2 is found to significantly contribute to the observed selectivity. The results reveal the basis for the potent and selective binding of VEGF-A{sub 164} to Nrp1.

  3. The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains*

    PubMed Central

    Dahlbäck, Madeleine; Jørgensen, Lars M.; Nielsen, Morten A.; Clausen, Thomas M.; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Arnot, David E.; Theander, Thor G.; Salanti, Ali

    2011-01-01

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. PMID:21398524

  4. 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

  5. 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. PMID:23953471

  6. 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.

  7. Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

    PubMed

    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 M A

    2016-02-18

    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

  8. Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

    PubMed Central

    Pozuelo Rubio, Mercedes; Campbell, David G.; Morrice, Nicholas A.; Mackintosh, Carol

    2005-01-01

    PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding. PMID:16153182

  9. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    PubMed

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

  10. 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

  11. The minor C-allele of rs2014355 in ACADS is associated with reduced insulin release following an oral glucose load

    PubMed Central

    2011-01-01

    Background A genome-wide association study (GWAS) using metabolite concentrations as proxies for enzymatic activity, suggested that two variants: rs2014355 in the gene encoding short-chain acyl-coenzyme A dehydrogenase (ACADS) and rs11161510 in the gene encoding medium-chain acyl-coenzyme A dehydrogenase (ACADM) impair fatty acid β-oxidation. Chronic exposure to fatty acids due to an impaired β-oxidation may down-regulate the glucose-stimulated insulin release and result in an increased risk of type 2 diabetes (T2D). We aimed to investigate whether the two variants associate with altered insulin release following an oral glucose load or with T2D. Methods The variants were genotyped using KASPar® PCR SNP genotyping system and investigated for associations with estimates of insulin release and insulin sensitivity following an oral glucose tolerance test (OGTT) in a random sample of middle-aged Danish individuals (n ACADS = 4,324; n ACADM = 4,337). The T2D-case-control study involved a total of ~8,300 Danish individuals (n ACADS = 8,313; n ACADM = 8,344). Results In glucose-tolerant individuals the minor C-allele of rs2014355 of ACADS associated with reduced measures of serum insulin at 30 min following an oral glucose load (per allele effect (β) = -3.8% (-6.3%;-1.3%), P = 0.003), reduced incremental area under the insulin curve (β = -3.6% (-6.3%;-0.9%), P = 0.009), reduced acute insulin response (β = -2.2% (-4.2%;0.2%), P = 0.03), and with increased insulin sensitivity ISIMatsuda (β = 2.9% (0.5%;5.2%), P = 0.02). The C-allele did not associate with two other measures of insulin sensitivity or with a derived disposition index. The C-allele was not associated with T2D in the case-control analysis (OR 1.07, 95% CI 0.96-1.18, P = 0.21). rs11161510 of ACADM did not associate with any indices of glucose-stimulated insulin release or with T2D. Conclusions In glucose-tolerant individuals the minor C-allele of rs2014355 of ACADS was associated with reduced measures of

  12. Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

    PubMed Central

    Priefert, H; Steinbüchel, A

    1992-01-01

    The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54. Images PMID:1356967

  13. Endoglycanase-Catalyzed Degradation of Hemicelluloses during Development of Carnation (Dianthus caryophyllus L.) Petals 1

    PubMed Central

    de Vetten, Nicolaas C.; Huber, Donald J.; Gross, Kenneth C.

    1991-01-01

    Large molecular-size hemicelluloses, including xyloglucan, decreased in quantity during development of carnation (Dianthus caryophyllus L. cv White Sim) petals, along with a relative increase in polymers with an average size of 10 kilodaltons. An enzyme extract from senescing petal tissue depolymerized the large molecular-size hemicelluloses in a pattern similar to that occurring in vivo during petal development. The products generated in vitro were composed of polymeric and monomeric components, the latter consisting primarily of xylose, galactose, and glucose. The 10 kilodalton hemicelluloses were resistant to in vitro enzymic hydrolysis. Glycosyl-linkage composition of the large molecular-size polymers provided evidence for the presence of xyloglucan with smaller amounts of arabinoxylan and arabinan. The 10 kilodalton polymers were enriched in mannosyl and 4-linked glucosyl residues, presumably derived from glucomannan. During petal development or enzymic hydrolysis, no change was observed in the relative glycosyl-linkage composition of the large molecular-size hemicelluloses. The in vitro activity of carnation petal enzymes active toward native hemicelluloses increased threefold at the onset of senescence and declined slightly thereafter. Gel chromatography revealed 23 and 12 kilodalton proteins with hemicellulase activity. The enzymes hydrolyzed the large molecular-size hemicelluloses extensively and without formation of monomers. Endoxylanase activity was detected in the partially purified enzyme preparation. Xyloglucan was depolymerized in the absence of cellulase activity, suggesting the presence of a xyloglucan-specific glucanase. These data indicate that the hemicellulose molecular-size changes observed during development of carnation petals are due, in part, to the enzymic depolymerization of large molecular-size hemicelluloses. PMID:16668064

  14. Potential roles of PINK1 for increased PGC-1α-mediated mitochondrial fatty acid oxidation and their associations with Alzheimer disease and diabetes

    PubMed Central

    Choi, Joungil; Ravipati, Avinash; Nimmagadda, Vamshi; Schubert, Manfred; Castellani, Rudolph J.; Russell, James W.

    2016-01-01

    Down-regulation of PINK1 and PGC-1α proteins is implicated in both mitochondrial dysfunction and oxidative stress potentially linking metabolic abnormality and neurodegeneration. Here, we report that PGC-1α and PINK1 expression is markedly decreased in Alzheimer disease (AD) and diabetic brains. We observed a significant down-regulation of PGC-1α and PINK1 protein expression in H2O2-treated cells but not in those cells treated with N-acetyl cysteine. The protein levels of two key enzymes of the mitochondrial β-oxidation machinery, acyl-coenzyme A dehydrogenase, very long chain (ACADVL) and mitochondrial trifunctional enzyme subunit α are significantly decreased in AD and diabetic brains. Moreover, we observed a positive relationship between ACADVL and 64 kDa PINK1 protein levels in AD and diabetic brains. Overexpression of PGC-1α decreases lipid-droplet accumulation and increases mitochondrial fatty acid oxidation; down-regulation of PINK1 abolishes these effects. Together, these results provide new insights into potential cooperative roles of PINK1 and PGC-1α in mitochondrial fatty acid oxidation, suggesting possible regulatory roles for mitochondrial function in the pathogenesis of AD and diabetes. PMID:25260493

  15. Arabidopsis Deficient in Cutin Ferulate Encodes a Transferase Required for Feruloylation of ω-Hydroxy Fatty Acids in Cutin Polyester1[W][OA

    PubMed Central

    Rautengarten, Carsten; Ebert, Berit; Ouellet, Mario; Nafisi, Majse; Baidoo, Edward E.K.; Benke, Peter; Stranne, Maria; Mukhopadhyay, Aindrila; Keasling, Jay D.; Sakuragi, Yumiko; Scheller, Henrik Vibe

    2012-01-01

    The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C16 and C18 unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm. PMID:22158675

  16. 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. PMID:26493158

  17. 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. PMID:22633641

  18. Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

    PubMed Central

    Wahlen, Bradley D.; Garner, EmmaLee; Wei, Jiashi; Seefeldt, Lance C.

    2012-01-01

    Wax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria: Marinobacter aquaeolei VT8, Acinetobacter baylyi, Rhodococcus jostii RHA1, and Psychrobacter cryohalolentis K5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. These in vitro results are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular from M. aquaeolei VT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production. PMID:22685145

  19. ABCG26-mediated polyketide trafficking and hydroxycinnamoyl spermidines contribute to pollen wall exine formation in Arabidopsis.

    PubMed

    Quilichini, Teagen D; Samuels, A Lacey; Douglas, Carl J

    2014-11-01

    Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26, which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5, POLYKETIDE SYNTHASE A [PKSA], PKSB, and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death. PMID:25415974

  20. ABCG26-Mediated Polyketide Trafficking and Hydroxycinnamoyl Spermidines Contribute to Pollen Wall Exine Formation in Arabidopsis[W

    PubMed Central

    Quilichini, Teagen D.; Samuels, A. Lacey; Douglas, Carl J.

    2014-01-01

    Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26, which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5, POLYKETIDE SYNTHASE A [PKSA], PKSB, and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death. PMID:25415974

  1. Plant fatty acyl reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications.

    PubMed

    Rowland, Owen; Domergue, Frédéric

    2012-09-01

    Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols. PMID:22794916

  2. LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase essential for pollen exine development in Arabidopsis.

    PubMed

    Dobritsa, Anna A; Lei, Zhentian; Nishikawa, Shuh-Ichi; Urbanczyk-Wochniak, Ewa; Huhman, David V; Preuss, Daphne; Sumner, Lloyd W

    2010-07-01

    Pollen grains of land plants have evolved remarkably strong outer walls referred to as exine that protect pollen and interact with female stigma cells. Exine is composed of sporopollenin, and while the composition and synthesis of this biopolymer are not well understood, both fatty acids and phenolics are likely components. Here, we describe mutations in the Arabidopsis (Arabidopsis thaliana) LESS ADHESIVE POLLEN (LAP5) and LAP6 that affect exine development. Mutation of either gene results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific proteins with homology to chalcone synthase, a key flavonoid biosynthesis enzyme. lap5 and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin. Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-coenzyme A yielded bis-noryangonin (a commonly reported derailment product of chalcone synthase), while similar in vitro analyses using fatty acyl-coenzyme A as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays indicate that LAP5 and LAP6 are multifunctional enzymes and may play a role in both the synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2) demonstrated that they act synergistically in exine production. PMID:20442277

  3. Characterization of enzymes involved in formation of ethyl esters of long-chain fatty acids in humans.

    PubMed

    Diczfalusy, M A; Björkhem, I; Einarsson, C; Hillebrant, C G; Alexson, S E

    2001-07-01

    Elevated fatty acid ethyl ester (FAEE) concentrations have been detected in postmortem organs from alcoholics and patients acutely intoxicated by alcohol, and FAEE have been implicated as mediators of ethanol-induced organ damage. The formation of FAEE is catalyzed by acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and by FAEE synthase, which utilize acyl-CoA and free fatty acids, respectively, as substrates. Because little is known about the capacity of various human tissues to synthesize and hydrolyze FAEE, we investigated formation of FAEE by AEAT and FAEE synthase in tissue homogenates from human gastric ventricular and duodenal mucosa, pancreas, liver, heart, lung, and adipose tissue, gallbladder mucosa, and in serum. Liver, duodenal mucosa, and pancreas were found to have the highest capacities to synthesize FAEE, mainly due to AEAT. FAEE hydrolyzing activity was highest in liver and pancreas, but hardly detectable in adipose tissue or heart. Because fatty acids and alcohol are absorbed by the intestinal mucosa, intestine may be a major site of FAEE synthesis, and FAEE may be delivered via the circulation to other organs and taken up by lipoprotein receptor-mediated uptake. A very low rate of FAEE hydrolysis was detected in heart and adipose tissue, which probably accounts for the previously observed accumulation of FAEE in these organs. PMID:11441128

  4. The Role of Lipotoxicity in Smoke Cardiomyopathy

    PubMed Central

    Santos, Priscila P.; Oliveira, Fernando; Ferreira, Vanessa C. M. P.; Polegato, Bertha F.; Roscani, Meliza G.; Fernandes, Ana Angelica; Modesto, Pamela; Rafacho, Bruna P. M.; Zanati, Silmeia G.; Di Lorenzo, Annarita; Matsubara, Luiz S.; Paiva, Sergio A. R.; Zornoff, Leonardo A. M.; Minicucci, Marcos F.; Azevedo, Paula S.

    2014-01-01

    Background/Aims Experimental and clinical studies have shown the direct toxic effects of cigarette smoke (CS) on the myocardium, independent of vascular effects. However, the underlying mechanisms are not well known. Methods Wistar rats were allocated to control (C) and cigarette smoke (CS) groups. CS rats were exposed to cigarette smoke for 2 months. Results After that morphometric, functional and biochemical parameters were measured. The echocardiographic study showed enlargement of the left atria, increase in the left ventricular systolic volume and reduced systolic function. Within the cardiac metabolism, exposure to CS decreased beta hydroxy acyl coenzyme A dehydrogenases and citrate synthases and increased lactate dehydrogenases. Peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were expressed similarly in both groups. CS increased serum lipids and myocardial triacylglycerols (TGs). These data suggest that impairment in fatty acid oxidation and the accumulation of cardiac lipids characterize lipotoxicity. CS group exhibited increased oxidative stress and decreased antioxidant defense. Finally, the myocyte cross-sectional area and active Caspase 3 were increased in the CS group. Conclusion The cardiac remodeling that was observed in the CS exposure model may be explained by abnormalities in energy metabolism, including lipotoxicity and oxidative stress. PMID:25462161

  5. 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. PMID:26303601

  6. Arabidopsis GPAT9 contributes to synthesis of intracellular glycerolipids but not surface lipids

    PubMed Central

    Singer, Stacy D.; Chen, Guanqun; Mietkiewska, Elzbieta; Tomasi, Pernell; Jayawardhane, Kethmi; Dyer, John M.; Weselake, Randall J.

    2016-01-01

    GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (GPAT) genes encode enzymes involved in glycerolipid biosynthesis in plants. Ten GPAT homologues have been identified in Arabidopsis. GPATs 4–8 have been shown to be involved in the production of extracellular lipid barrier polyesters. Recently, GPAT9 was reported to be essential for triacylglycerol (TAG) biosynthesis in developing Arabidopsis seeds. The enzymatic properties and possible functions of GPAT9 in surface lipid, polar lipid and TAG biosynthesis in non-seed organs, however, have not been investigated. Here we show that Arabidopsis GPAT9 exhibits sn-1 acyltransferase activity with high specificity for acyl-coenzyme A, thus providing further evidence that this GPAT is involved in storage lipid biosynthesis. We also confirm a role for GPAT9 in seed oil biosynthesis and further demonstrate that GPAT9 contributes to the biosynthesis of both polar lipids and TAG in developing leaves, as well as lipid droplet production in developing pollen grains. Conversely, alteration of constitutive GPAT9 expression had no obvious effects on surface lipid biosynthesis. Taken together, these studies expand our understanding of GPAT9 function to include modulation of several different intracellular glycerolipid pools in plant cells. PMID:27325892

  7. A novel class of antihyperlipidemic agents with low density lipoprotein receptor up-regulation via the adaptor protein autosomal recessive hypercholesterolemia.

    PubMed

    Asano, Shigehiro; Ban, Hitoshi; Tsuboya, Norie; Uno, Shinsaku; Kino, Kouichi; Ioriya, Katsuhisa; Kitano, Masafumi; Ueno, Yoshihide

    2010-04-22

    We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this study we focused on compound 2, a unique LDL-R up-regulator, and describe the discovery of a novel class of up-regulators of LDL-R. Replacement the methylene urea linker in compound 2 with an acylsulfonamide linker kept a potent LDL-R up-regulatory activity, and subsequent optimization work gave compound 39 as a highly potent LDL-R up-regulator (39; EC(25) = 0.047 microM). Compound 39 showed no ACAT inhibitory activity even at 1 microM. The sodium salts of compound 39 reduced plasma total and LDL cholesterol levels in a dose-dependent manner in an experimental animal model of hyperlipidemia. Moreover, we revealed in this study using RNA interference that autosomal recessive hypercholesterolemia (ARH), an adaptor protein of LDL-R, is essential for compound 39 up-regulation of LDL-R expression. PMID:20356098

  8. Arabidopsis GPAT9 contributes to synthesis of intracellular glycerolipids but not surface lipids.

    PubMed

    Singer, Stacy D; Chen, Guanqun; Mietkiewska, Elzbieta; Tomasi, Pernell; Jayawardhane, Kethmi; Dyer, John M; Weselake, Randall J

    2016-08-01

    GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (GPAT) genes encode enzymes involved in glycerolipid biosynthesis in plants. Ten GPAT homologues have been identified in Arabidopsis. GPATs 4-8 have been shown to be involved in the production of extracellular lipid barrier polyesters. Recently, GPAT9 was reported to be essential for triacylglycerol (TAG) biosynthesis in developing Arabidopsis seeds. The enzymatic properties and possible functions of GPAT9 in surface lipid, polar lipid and TAG biosynthesis in non-seed organs, however, have not been investigated. Here we show that Arabidopsis GPAT9 exhibits sn-1 acyltransferase activity with high specificity for acyl-coenzyme A, thus providing further evidence that this GPAT is involved in storage lipid biosynthesis. We also confirm a role for GPAT9 in seed oil biosynthesis and further demonstrate that GPAT9 contributes to the biosynthesis of both polar lipids and TAG in developing leaves, as well as lipid droplet production in developing pollen grains. Conversely, alteration of constitutive GPAT9 expression had no obvious effects on surface lipid biosynthesis. Taken together, these studies expand our understanding of GPAT9 function to include modulation of several different intracellular glycerolipid pools in plant cells. PMID:27325892

  9. 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

    2016-08-01

    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. PMID:27477280

  10. Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

    PubMed Central

    van der Vlugt-Bergmans, Cécile J. B; van der Werf, Mariët J.

    2001-01-01

    A monoterpene ɛ-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism. PMID:11157238

  11. Distinct Structural Elements Dictate the Specificity of the Type III Pentaketide Synthase from Neurospora crassa

    SciTech Connect

    Rubin-Pitel, Sheryl B.; Zhang, Houjin; Vu, Trang; Brunzelle, Joseph S.; Zhao, Huimin; Nair, Satish K.

    2009-01-15

    The fungal type III polyketide synthase 2'-oxoalkylresorcyclic acid synthase (ORAS) primes with a range of acyl-Coenzyme A thioesters (C{sub 4}--C{sub 20}) and extends using malonyl-Coenzyme A to produce pyrones, resorcinols, and resorcylic acids. To gain insight into this unusual substrate specificity and product profile, we have determined the crystal structures of ORAS to 1.75 {angstrom} resolution, the Phe-252{yields}Gly site-directed mutant to 2.1 {angstrom} resolution, and a binary conplex of ORAS with eicosanoic acid to 2.0 {angstrom} resolution. The structures reveal a distinct rearrangement of structural elements near the active site that allows accomodation of long-chain fatty acid esters and a reorientation of the gating mechanism that controls cyclization and polyketide chain length. The roles of these structural elements are further elucidated by characterization of various structure-based site-directed variants. These studies establish an unexpected plasticity to the PKS fold, unanticipated from structural studies of other members of this enzyme family.

  12. Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans.

    PubMed

    Hagmann, Anna; Hunkeler, Moritz; Stuttfeld, Edward; Maier, Timm

    2016-08-01

    Biotin-dependent acyl-coenzyme A (CoA) carboxylases (aCCs) are involved in key steps of anabolic pathways and comprise three distinct functional units: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyl transferase (CT). YCC multienzymes are a poorly characterized family of prokaryotic aCCs of unidentified substrate specificity, which integrate all functional units into a single polypeptide chain. We employed a hybrid approach to study the dynamic structure of Deinococcus radiodurans (Dra) YCC: crystal structures of isolated domains reveal a hexameric CT core with extended substrate binding pocket and a dimeric BC domain. Negative-stain electron microscopy provides an approximation of the variable positioning of the BC dimers relative to the CT core. Small-angle X-ray scattering yields quantitative information on the ensemble of Dra YCC structures in solution. Comparison with other carrier protein-dependent multienzymes highlights a characteristic range of large-scale interdomain flexibility in this important class of biosynthetic enzymes. PMID:27396827

  13. CLI-095 decreases atherosclerosis by modulating foam cell formation in apolipoprotein E-deficient mice.

    PubMed

    Wang, Xiao-Qing; Wan, Hui-Qing; Wei, Xian-Jing; Zhang, Ying; Qu, Peng

    2016-07-01

    Toll-like receptor 4 (TLR4) is considered to have a critical role in the occurrence and development of atherosclerosis in atherosclerosis-prone mice; however, it remains uncertain whether treatment with a TLR4 inhibitor may attenuate atherosclerosis. The present study aimed to determine the vascular protective effects of the TLR4 inhibitor CLI-095 on apolipoprotein E‑deficient (ApoE‑/‑) mice. ApoE‑/‑ mice were fed either chow or a high‑fat diet, and were treated with or without CLI‑095 for 10 weeks. The mean atherosclerotic plaque area in the aortic sections of CLI‑095‑treated mice was 54.3% smaller than in the vehicle‑treated mice (P=0.0051). In vitro, murine peritoneal macrophages were treated with or without CLI‑095, and were subsequently stimulated with oxidized low‑density lipoprotein. Treatment with CLI‑095 markedly reduced the expression levels of lectin‑like oxidized low‑density lipoprotein receptor‑1 and acyl-coenzyme A:cholesterol acyltransferase‑1, and significantly upregulated the expression levels of ATP‑binding cassette transporter A1, predominantly via suppressing activation of the TLR4/nuclear factor‑κB signaling pathway. The results of the present study indicated that the TLR4 inhibitor CLI‑095 has the ability to suppress the progression of atherosclerosis in an in vivo model by reducing macrophage foam cell formation. PMID:27176130

  14. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT).

    PubMed

    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

  15. CLI-095 decreases atherosclerosis by modulating foam cell formation in apolipoprotein E-deficient mice

    PubMed Central

    WANG, XIAO-QING; WAN, HUI-QING; WEI, XIAN-JING; ZHANG, YING; QU, PENG

    2016-01-01

    Toll-like receptor 4 (TLR4) is considered to have a critical role in the occurrence and development of atherosclerosis in atherosclerosis-prone mice; however, it remains uncertain whether treatment with a TLR4 inhibitor may attenuate atherosclerosis. The present study aimed to determine the vascular protective effects of the TLR4 inhibitor CLI-095 on apolipoprotein E-deficient (ApoE−/−) mice. ApoE−/− mice were fed either chow or a high-fat diet, and were treated with or without CLI-095 for 10 weeks. The mean atherosclerotic plaque area in the aortic sections of CLI-095-treated mice was 54.3% smaller than in the vehicle-treated mice (P=0.0051). In vitro, murine peritoneal macrophages were treated with or without CLI-095, and were subsequently stimulated with oxidized low-density lipoprotein. Treatment with CLI-095 markedly reduced the expression levels of lectin-like oxidized low-density lipoprotein receptor-1 and acyl-coenzyme A:cholesterol acyltransferase-1, and significantly upregulated the expression levels of ATP-binding cassette transporter A1, predominantly via suppressing activation of the TLR4/nuclear factor-κB signaling pathway. The results of the present study indicated that the TLR4 inhibitor CLI-095 has the ability to suppress the progression of atherosclerosis in an in vivo model by reducing macrophage foam cell formation. PMID:27176130

  16. Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota

    PubMed Central

    Polansky, Ondrej; Sekelova, Zuzana; Faldynova, Marcela; Sebkova, Alena; Sisak, Frantisek

    2015-01-01

    The gut microbiota plays important roles in its host. However, how each microbiota member contributes to the behavior of the whole population is not known. In this study, we therefore determined protein expression in the cecal microbiota in chickens of selected ages and in 7-day-old chickens inoculated with different cecal extracts on the day of hatching. Campylobacter, Helicobacter, Mucispirillum, and Megamonas overgrew in the ceca of 7-day-old chickens inoculated with cecal extracts from donor hens. Firmicutes were characterized by ABC and phosphotransferase system (PTS) transporters, extensive acyl coenzyme A (acyl-CoA) metabolism, and expression of l-fucose isomerase. Anaerostipes, Anaerotruncus, Pseudoflavonifractor, Dorea, Blautia, and Subdoligranulum expressed spore proteins. Firmicutes (Faecalibacterium, Butyrivibrio, Megasphaera, Subdoligranulum, Oscillibacter, Anaerostipes, and Anaerotruncus) expressed enzymes required for butyrate production. Megamonas, Phascolarctobacterium, and Blautia (exceptions from the phylum Firmicutes) and all Bacteroidetes expressed enzymes for propionate production pathways. Representatives of Bacteroidetes also expressed xylose isomerase, enzymes required for polysaccharide degradation, and ExbBD, TonB, and outer membrane receptors likely to be involved in oligosaccharide transport. Based on our data, Anaerostipes, Anaerotruncus, and Subdoligranulum might be optimal probiotic strains, since these represent spore-forming butyrate producers. However, certain care should be taken during microbiota transplantation because the microbiota may behave differently in the intestinal tract of a recipient depending on how well the existing communities are established. PMID:26712550

  17. ABI4 Activates DGAT1 Expression in Arabidopsis Seedlings during Nitrogen Deficiency1[C][W][OA

    PubMed Central

    Yang, Yang; Yu, Xiangchun; Song, Lianfen; An, Chengcai

    2011-01-01

    Triacylglycerol (TAG) is the major seed storage lipid and is important for biofuel and other renewable chemical uses. Acyl-coenzyme A:diacylglycerol acyltransferase1 (DGAT1) is the rate-limiting enzyme in the TAG biosynthesis pathway, but the mechanism of its regulation is unknown. Here, we show that TAG accumulation in Arabidopsis (Arabidopsis thaliana) seedlings increased significantly during nitrogen deprivation (0.1 mm nitrogen) with concomitant induction of genes involved in TAG biosynthesis and accumulation, such as DGAT1 and OLEOSIN1. Nitrogen-deficient seedlings were used to determine the key factors contributing to ectopic TAG accumulation in vegetative tissues. Under low-nitrogen conditions, the phytohormone abscisic acid plays a crucial role in promoting TAG accumulation in Arabidopsis seedlings. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that ABSCISIC ACID INSENSITIVE4 (ABI4), an important transcriptional factor in the abscisic acid signaling pathway, bound directly to the CE1-like elements (CACCG) present in DGAT1 promoters. Genetic studies also revealed that TAG accumulation and DGAT1 expression were reduced in the abi4 mutant. Taken together, our results indicate that abscisic acid signaling is part of the regulatory machinery governing TAG ectopic accumulation and that ABI4 is essential for the activation of DGAT1 in Arabidopsis seedlings during nitrogen deficiency. PMID:21515696

  18. Evidence for a complex of three beta-oxidation enzymes in Escherichia coli: induction and localization.

    PubMed Central

    O'Brien, W J; Frerman, F E

    1977-01-01

    The enzymes for beta-oxidation of fatty acids in inducible and constitutive strains of Escherichia coli were assayed in soluble and membrane fractions of disrupted cells by using fatty acid and acyl-coenzyme A (CoA) substrates containing either 4 or 16 carbon atoms in the acyl moieties. Cell fractionation was monitored, using succinic dehydrogenase as a membrane marker and glucose 6-phosphate dehydrogenase as a soluble marker. Acyl-CoA synthetase activity was detected exclusively in the membrane fraction, whereas acyl-CoA dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities that utilized both C4 and C16 acyl-CoA substrates were isolated from the soluble fraction. 3-Hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase activities assayed with both C4 and C16 acyl-CoA substrates co-chromatographed on gel filtration and ion-exchange columns and cosedimented in glycerol gradients. The data show that these three enzyme activities of the fad regulon can be isolated as a multienzyme complex. This complex dissociates in very dilute preparations; however, in those preparations where the three activities are separated, the fractionated species retain activity with both C4 and C16 acyl-CoA substrates. PMID:334745

  19. VY6, a β-lactoglobulin-derived peptide, altered metabolic lipid pathways in the zebra fish liver.

    PubMed

    Mohammed-Geba, K; Arrutia, F; Do-Huu, H; Borrell, Y J; Galal-Khallaf, A; Ardura, A; Riera, Francisco A; Garcia-Vazquez, Eva

    2016-04-20

    Today enormous research efforts are being focused on alleviating the massive, adverse effects of obesity. Short peptides are key targets for research as they can be generated from natural proteins, like milk. Here we conducted trypsinogen digestion of beta-lactoglobulin (β-lg), the major mammalian milk protein, to release the hexamer VY6. It was assayed in vivo for its activities on lipid metabolism using zebra fish as a vertebrate model. Zebra fish juveniles were injected with two different doses of the peptide: 100 and 800 μg per g fish and left for 5 days before sacrificing. Lipid measurements showed significant reduction in liver triglycerides and free cholesterol, as well as increased liver HDL cholesterol. Dose-dependent increases of the mRNA levels of the genes coding for the enzymes acyl coenzyme A oxidase 1 (acox1) and lipoprotein lipase (lpl) were also found. The complete results suggest significant anti-obesity activity of the β-lg-derived VY6 peptide. Its use as a nutraceutical has been discussed. PMID:26983953

  20. The effect of chronic exposure to high palmitic acid concentrations on the aerobic metabolism of human endothelial EA.hy926 cells.

    PubMed

    Broniarek, Izabela; Koziel, Agnieszka; Jarmuszkiewicz, Wieslawa

    2016-09-01

    A chronic elevation of circulating free fatty acids (FFAs) is associated with diseases like obesity or diabetes and can lead to lipotoxicity. The goals of this study were to assess the influence of chronic exposure to high palmitic acid (PAL) levels on mitochondrial respiratory functions in endothelial cells and isolated mitochondria. Human umbilical vein endothelial cells (EA.hy926 line) were grown for 6 days in a medium containing either 100 or 150 μM PAL. Growth at high PAL concentrations induced a considerable increase in fatty acid-supplied respiration and a reduction of mitochondrial respiration during carbohydrate and glutamine oxidation. High PAL levels elevated intracellular and mitochondrial superoxide generation; increased inflammation marker, acyl-coenzyme A (CoA) dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression; and decreased hexokinase I and pyruvate dehydrogenase expression. No change in aerobic respiration capacity was observed, while fermentation was decreased. In mitochondria isolated from high PAL-treated cells, an increase in the oxidation of palmitoylcarnitine, a decrease in the oxidation of pyruvate, and an increase in UCP2 activity were observed. Our results demonstrate that exposure to high PAL levels induces a shift in endothelial aerobic metabolism toward the oxidation of fatty acids. Increased levels of PAL caused impairment and uncoupling of the mitochondrial oxidative phosphorylation system. Our data indicate that FFAs significantly affect endothelial oxidative metabolism, reactive oxygen species (ROS) formation, and cell viability and, thus, might contribute to endothelial and vascular dysfunction. PMID:27417103

  1. 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

  2. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

    PubMed Central

    Haas, A; Wickner, W

    1996-01-01

    In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p. Images PMID:8670830

  3. 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. PMID:20425236

  4. Important Metabolic Pathways and Biological Processes Expressed by Chicken Cecal Microbiota.

    PubMed

    Polansky, Ondrej; Sekelova, Zuzana; Faldynova, Marcela; Sebkova, Alena; Sisak, Frantisek; Rychlik, Ivan

    2016-03-01

    The gut microbiota plays important roles in its host. However, how each microbiota member contributes to the behavior of the whole population is not known. In this study, we therefore determined protein expression in the cecal microbiota in chickens of selected ages and in 7-day-old chickens inoculated with different cecal extracts on the day of hatching. Campylobacter, Helicobacter, Mucispirillum, and Megamonas overgrew in the ceca of 7-day-old chickens inoculated with cecal extracts from donor hens. Firmicutes were characterized by ABC and phosphotransferase system (PTS) transporters, extensive acyl coenzyme A (acyl-CoA) metabolism, and expression of l-fucose isomerase. Anaerostipes, Anaerotruncus, Pseudoflavonifractor, Dorea, Blautia, and Subdoligranulum expressed spore proteins. Firmicutes (Faecalibacterium, Butyrivibrio, Megasphaera, Subdoligranulum, Oscillibacter, Anaerostipes, and Anaerotruncus) expressed enzymes required for butyrate production. Megamonas, Phascolarctobacterium, and Blautia (exceptions from the phylum Firmicutes) and all Bacteroidetes expressed enzymes for propionate production pathways. Representatives of Bacteroidetes also expressed xylose isomerase, enzymes required for polysaccharide degradation, and ExbBD, TonB, and outer membrane receptors likely to be involved in oligosaccharide transport. Based on our data, Anaerostipes, Anaerotruncus, and Subdoligranulum might be optimal probiotic strains, since these represent spore-forming butyrate producers. However, certain care should be taken during microbiota transplantation because the microbiota may behave differently in the intestinal tract of a recipient depending on how well the existing communities are established. PMID:26712550

  5. Rimonabant is a dual inhibitor of acyl CoA:cholesterol acyltransferases 1 and 2.

    PubMed

    Netherland, Courtney; Thewke, Douglas P

    2010-08-01

    Acyl coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannabinoid receptor antagonists, AM251 and SR144528, recently identified as potent inhibitors of ACAT. Therefore, we examined the effects of Rimonabant on ACAT using both in vivo cell-based assays and in vitro cell-free assays. Rimonabant dose-dependently reduced ACAT activity in Raw 264.7 macrophages (IC(50)=2.9+/-0.38 microM) and isolated peritoneal macrophages. Rimonabant inhibited ACAT activity in intact CHO-ACAT1 and CHO-ACAT2 cells and in cell-free assays with approximately equal efficiency (IC(50)=1.5+/-1.2 microM and 2.2+/-1.1 microM for CHO-ACAT1 and CHO-ACAT2, respectively). Consistent with ACAT inhibition, Rimonabant treatment blocked ACAT-dependent processes in macrophages, oxysterol-induced apoptosis and acetylated-LDL induced foam cell formation. From these results we conclude that Rimonabant is an ACAT1/2 dual inhibitor and suggest that some of the atherosclerotic beneficial effects of Rimonabant are, at least partly, due to inhibition of ACAT. PMID:20609360

  6. 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

  7. Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB

    PubMed Central

    Hamill, Michael J.; Jost, Marco; Wong, Cintyu; Bene, Nicholas C.; Drennan, Catherine L.; Elliott, Sean J.

    2012-01-01

    When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB) in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD). In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at −181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD) family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes. PMID:23443126

  8. Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling

    SciTech Connect

    Pollard, M.; Ohlrogge, J.

    1999-12-01

    Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [{sup 13}C{sub 2}{sup 18}O{sub 2}]Acetate was incubated with spinach (Spinacia oleracea) leaves and the {sup 18}O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectrometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an {sup 18}O 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 {sup 18}O 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 {sup 18}O or, less likely, complete loss of {sup 18}O, but not a 50% loss of {sup 18}O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of prokaryotic and eukaryotic lipids have both been confirmed.

  9. High throughput de novo RNA sequencing elucidates novel responses in Penicillium chrysogenum under microgravity.

    PubMed

    Sathishkumar, Yesupatham; Krishnaraj, Chandran; Rajagopal, Kalyanaraman; Sen, Dwaipayan; Lee, Yang Soo

    2016-02-01

    In this study, the transcriptional alterations in Penicillium chrysogenum under simulated microgravity conditions were analyzed for the first time using an RNA-Seq method. The increasing plethora of eukaryotic microbial flora inside the spaceship demands the basic understanding of fungal biology in the absence of gravity vector. Penicillium species are second most dominant fungal contaminant in International Space Station. Penicillium chrysogenum an industrially important organism also has the potential to emerge as an opportunistic pathogen for the astronauts during the long-term space missions. But till date, the cellular mechanisms underlying the survival and adaptation of Penicillium chrysogenum to microgravity conditions are not clearly elucidated. A reference genome for Penicillium chrysogenum is not yet available in the NCBI database. Hence, we performed comparative de novo transcriptome analysis of Penicillium chrysogenum grown under microgravity versus normal gravity. In addition, the changes due to microgravity are documented at the molecular level. Increased response to the environmental stimulus, changes in the cell wall component ABC transporter/MFS transporters are noteworthy. Interestingly, sustained increase in the expression of Acyl-coenzyme A: isopenicillin N acyltransferase (Acyltransferase) under microgravity revealed the significance of gravity in the penicillin production which could be exploited industrially. PMID:26603994

  10. Evaluation of two novel biochemicals on plasma and egg yolk lipid composition and laying hen performance.

    PubMed

    Elkin, R G; Freed, M; Watkins, B A; Srebnik, M; Kieft, K A; Newton, R S

    1993-03-01

    PD132301-2, an inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT; EC 2.3.1.26), and 1-stearylboronic acid (SBA), a fatty acid analogue, were orally administered to White Leghorn hens in separate experiments to evaluate their effects on layer performance and plasma and egg yolk lipids. Five 60-wk-old hens each were fed either a corn-soybean meal basal layer ration, or the basal diet supplemented with .0121, .0363, or .1089% PD132301-2. In a second experiment, 12 37-wk-old hens each were fed either a basal layer ration, or the basal diet supplemented with .20 or .40% SBA. The duration of the experiments were 21 and 16 days, respectively. Neither compound significantly affected hen-day production, egg weight, yolk weight, BW gain, feed consumption, feed efficiency, plasma total cholesterol and triglyceride concentrations, or egg yolk cholesterol content. PD132301-2 had no effect on yolk fatty acid profiles, and C22:6n3 was the only fatty acid altered by SBA. Although 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been successful in reducing egg cholesterol, ACAT inhibitors and fatty acid analogues apparently hold little promise in this regard. The results of the present work also support the concept that, in order to pharmacologically alter the cholesterol content of eggs, direct inhibition of key enzymes in the cholesterol biosynthetic pathway is necessary. PMID:8464792

  11. Effects of Choline on DNA Methylation and Macronutrient Metabolic Gene Expression in In Vitro Models of Hyperglycemia

    PubMed Central

    Jiang, Xinyin; Greenwald, Esther; Jack-Roberts, Chauntelle

    2016-01-01

    Choline is an essential nutrient that plays an important role in lipid metabolism and DNA methylation. Studies in rodents suggest that choline may adversely affect glycemic control, yet studies in humans are lacking. Using the human hepatic and placental cells, HepG2 and BeWo, respectively, we examined the interaction between choline and glucose treatments. In HepG2 cells, choline supplementation (1 mM) increased global DNA methylation and DNA methyltransferase expression in both low-glucose (5 mM) and high-glucose (35 mM) conditions. Choline supplementation increased the expression of peroxisomal acyl-coenzyme A oxidase 1 (ACOX1), which mediates fatty acid β-oxidation, especially in the high-glucose condition. High-glucose exposure increased the transcription of the gluconeogenic gene phosphoenolpyruvate carboxykinase (PEPCK), while choline supplementation mitigated such increase. Compared to HepG2 cells, the placenta-derived BeWo cells were relatively unresponsive to either high-glucose or -choline treatment. In conclusion, choline and glucose interacted to affect macronutrient metabolic genes, yet there was no indication that choline may worsen glycemic control in these in vitro human cell culture models. PMID:27081315

  12. Gene-Specific Involvement of β-Oxidation in Wound-Activated Responses in Arabidopsis1

    PubMed Central

    Castillo, M. Cruz; Martínez, Cristina; Buchala, Antony; Métraux, Jean-Pierre; León, José

    2004-01-01

    The coordinated induced expression of β-oxidation genes is essential to provide the energy supply for germination and postgerminative development. However, very little is known about other functions of β-oxidation in nonreserve organs. We have identified a gene-specific pattern of induced β-oxidation gene expression in wounded leaves of Arabidopsis. Mechanical damage triggered the local and systemic induction of only ACX1 among acyl-coenzyme A oxidase (ACX) genes, and KAT2/PED1 among 3-ketoacyl-coenzyme A thiolase (KAT) genes in Arabidopsis. In turn, wounding induced KAT5/PKT2 only systemically. Although most of the β-oxidation genes were activated by wound-related factors such as dehydration and abscisic acid, jasmonic acid (JA) induced only ACX1 and KAT5. Reduced expression of ACX1 or KAT2 genes, in transgenic plants expressing their corresponding mRNAs in antisense orientation, correlated with defective wound-activated synthesis of JA and with reduced expression of JA-responsive genes. Induced expression of JA-responsive genes by exogenous application of JA was unaffected in those transgenic plants, suggesting that ACX1 and KAT2 play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded Arabidopsis plants. PMID:15141068

  13. Engineering the filamentous fungus Neurospora crassa for lipid production from lignocellulosic biomass.

    PubMed

    Roche, Christine M; Glass, N Louise; Blanch, Harvey W; Clark, Douglas S

    2014-06-01

    Microbially produced triacylglycerol (TAG) is a potential feedstock for the production of biodiesel, but its commercialization will require high yields from low-cost renewable feedstocks such as lignocellulose. The present study employs a multi-gene approach to increasing TAG biosynthesis in the filamentous fungus Neurospora crassa. We demonstrate the redirection of carbon flux from glycogen biosynthesis towards fatty acid biosynthesis in a glycogen synthase deletion strain (Δgsy-1). Furthermore, combining Δgsy-1 with an enhanced TAG biosynthetic strain (acyl-Coenzyme A synthase; Δacs-3) of N. crassa yielded a twofold increase in total fatty acid accumulation over the control strain. The cellulose degrading potential of this double deletion strain was improved by deleting of the carbon catabolite regulation transcription factor (Δcre-1) to create the triple deletion strain Δacs-3 Δcre-1; Δgsy-1. This strain exhibited early and increased cellulase expression, as well as fourfold increased total fatty acid accumulation over the control on inhibitor-free model cellulose medium. The Δcre-1 mutation, however, was not beneficial for total fatty acid accumulation from pretreated lignocellulose. Conversion of dilute-acid pretreated Miscanthus to TAG was maximum in the constructed strain Δacs-3; Δgsy-1, which accumulated 2.3-fold more total fatty acid than the wild-type control strain, corresponding to a total fatty acid yield of 37.9 mg/g dry untreated Miscanthus. PMID:24700367

  14. Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling

    PubMed Central

    Ko, Hyun-Suk; Lee, Hyo-Jeong; Lee, Hyo-Jung; Sohn, Eun Jung; Yun, Miyong; Lee, Min-Ho; Kim, Sung-Hoon

    2013-01-01

    Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPARγ and CEBPα in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPARγ during adipocyte differentiation. Furthermore, PPARγ inhibitor GW9662 enhanced the decreased expression of FABP and PPARγ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPARγ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPARγ-related signaling. PMID:23997801

  15. Orthogonal Fatty Acid Biosynthetic Pathway Improves Fatty Acid Ethyl Ester Production in Saccharomyces cerevisiae.

    PubMed

    Eriksen, Dawn T; HamediRad, Mohammad; Yuan, Yongbo; Zhao, Huimin

    2015-07-17

    Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms. PMID:25594225

  16. Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli.

    SciTech Connect

    Lennen, Rebecca M.; Kruziki, Max A.; Kumar, Kritika; Zinkel, Robert A.; Burnum, Kristin E.; Lipton, Mary S.; Hoover, Spencer W.; Ranatunga, Don Ruwan; Wittkopp, Tyler M.; Marner II, Wesley D.; Pfleger, Brian F.

    2011-11-01

    Microbially produced fatty acids are potential precursors to high energy density biofuels, including alkanes and alkyl ethyl esters by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversions of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium chain length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long chain unsaturated fatty acid content greatly increased and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability, however little to no change in FFA titers was observed after 24 h cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.

  17. Altering small and medium alcohol selectivity in the wax ester synthase.

    PubMed

    Barney, Brett M; Ohlert, Janet M; Timler, Jacobe G; Lijewski, Amelia M

    2015-11-01

    The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or wax ester synthase) catalyzes the terminal reaction in the bacterial wax ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. The wild-type wax ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the wax ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for wax ester synthase, which will aid rational approaches to improve this enzyme with specific substrates. PMID:26205519

  18. 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

  19. Intermediate Energy Metabolism of Leptospira

    PubMed Central

    Baseman, J. B.; Cox, C. D.

    1969-01-01

    Metabolic studies were performed on three representative serotypes of Leptospira: a water isolate designated B16 and two pathogenic serotypes, pomona and schueffneri. Examination of whole cells of B16 for their ability to oxidize various substrates revealed that oleate significantly stimulated oxygen uptake. The respiratory quotient of 0.7 implied that oleate was degraded to carbon dioxide and water. Other substrates, such as carbohydrates, alcohols, intermediates of the citric acid cycle, and short-chain acids, including selected amino acids, did not stimulate endogenous respiration of whole cells. No oxygen uptake could be measured when cell-free extracts were tested with the substrates used with whole cells. Enzymatic analyses of cell-free extracts of the three strains demonstrated enzymes of the citric acid cycle, enzymes of the glycolytic and pentose pathways, and the general acyl coenzyme A dehydrogenase required for β-oxidation of fatty acids. Strain B16 and the two pathogenic serotypes appeared to possess similar metabolic capabilities. Enzymatic data might also explain the apparent inability of B16 to oxidize other substrates; kinases necessary for activation of common nonphosphorylated compounds were not detected in leptospiral extracts. These findings emphasized the dependence of leptospiral growth upon long-chain fatty acids. PMID:5776541

  20. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  1. Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket.

    PubMed Central

    Engel, C K; Mathieu, M; Zeelen, J P; Hiltunen, J K; Wierenga, R K

    1996-01-01

    The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates. Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex. The hexamer is a dimer of trimers. The monomer is folded into a right-handed spiral of four turns, followed by two small domains which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It confirms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importance of Glu144 as the catalytic base for the activation of a water molecule in the hydratase reaction. The comparison of an unliganded and a liganded active site within the same crystal form shows a water molecule in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water during catalysis. Images PMID:8895557

  2. Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins.

    PubMed

    Schmid, Manfred; Poulsen, Mathias Bach; Olszewski, Pawel; Pelechano, Vicent; Saguez, Cyril; Gupta, Ishaan; Steinmetz, Lars M; Moore, Claire; Jensen, Torben Heick

    2012-07-27

    Poly(A) (pA) tail binding proteins (PABPs) control mRNA polyadenylation, stability, and translation. In a purified system, S. cerevisiae PABPs, Pab1p and Nab2p, are individually sufficient to provide normal pA tail length. However, it is unknown how this occurs in more complex environments. Here we find that the nuclear exosome subunit Rrp6p counteracts the in vitro and in vivo extension of mature pA tails by the noncanonical pA polymerase Trf4p. Moreover, PABP loading onto nascent pA tails is controlled by Rrp6p; while Pab1p is the major PABP, Nab2p only associates in the absence of Rrp6p. This is because Rrp6p can interact with Nab2p and displace it from pA tails, potentially leading to RNA turnover, as evidenced for certain pre-mRNAs. We suggest that a nuclear mRNP surveillance step involves targeting of Rrp6p by Nab2p-bound pA-tailed RNPs and that pre-mRNA abundance is regulated at this level. PMID:22683267

  3. Analysis of human follistatin structure: identification of two discontinuous N-terminal sequences coding for activin A binding and structural consequences of activin binding to native proteins.

    PubMed

    Wang, Q; Keutmann, H T; Schneyer, A L; Sluss, P M

    2000-09-01

    A primary physiological function of follistatin is the binding and neutralization of activin, a transforming growth factor-beta family growth factor, and loss of function mutations are lethal. Despite the critical biological importance of follistatin's neutralization of activin, the structural basis of activin's binding to follistatin is poorly understood. The purposes of these studies were 1) to identify the primary sequence(s) within the N-terminal domain of the follistatin coding for activin binding, and 2) to determine whether activin binding to the native protein causes changes in other structural domains of follistatin. Synthetic peptide mimotopes identified within a 63-residue N-terminal domain two discontinuous sequences capable of binding labeled activin A. The first is located in a region (amino acids 3-26) of follistatin, a site previously identified by directed mutagenesis as important for activin binding. The second epitope, predicted to be located between amino acids 46 and 59, is newly identified. Although the sequences 3-26 and 46-59 code for activin binding, native follistatin only binds activin if disulfide bonding is intact. Furthermore, pyridylethylation of Cys residues followed by N-terminal sequencing and amino acid analysis revealed that all of the Cys residues in follistatin are involved in disulfide bonds and lack reactive free sulfhydryl groups. Specific ligands were used to probe the structural effects of activin binding on the other domains of the full-length molecule, comprised largely of the three 10-Cys follistatin module domains. No effects on ligand binding to follistatin-like module I or II were observed after the binding of activin A to native protein. In contrast, activin binding diminished recognition of domain III and enhanced that of the C domain by their respective monoclonal antibody probes, indicating an alteration of the antigenic structures of these regions. Thus, subsequent to activin binding, interactions are likely to occur between regions of follistatin located in different domains and separated by considerable lengths of linear sequence. Such interactions could have important functional significance with respect to the structural heterogeneity of naturally occurring follistatins. PMID:10965889

  4. Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity*♦

    PubMed Central

    Visweswaraiah, Jyothsna; Lageix, Sebastien; Castilho, Beatriz A.; Izotova, Lara; Kinzy, Terri Goss; Hinnebusch, Alan G.; Sattlegger, Evelyn

    2011-01-01

    The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His6-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His6-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. PMID:21849502

  5. Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity.

    PubMed

    Visweswaraiah, Jyothsna; Lageix, Sebastien; Castilho, Beatriz A; Izotova, Lara; Kinzy, Terri Goss; Hinnebusch, Alan G; Sattlegger, Evelyn

    2011-10-21

    The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. PMID:21849502

  6. Selective sampling and measurement of Cr (VI) in water with polyquaternary ammonium salt as a binding agent in diffusive gradients in thin-films technique.

    PubMed

    Chen, Hong; Zhang, Yang-Yang; Zhong, Ke-Li; Guo, Lian-Wen; Gu, Jia-Li; Bo, Le; Zhang, Meng-Han; Li, Jian-Rong

    2014-04-30

    A diffusive gradients in thin films (DGT) device with polyquaternary ammonium salt (PQAS) as a novel binding agent (PQAS DGT) combined with graphite furnace atomic absorption spectrometry (GFAAS) was developed for the selective sampling and measurement of Cr (VI) in water. The performance of PQAS DGT was independent of pH 3-12 and ionic strength from 1 × 10(-3) to 1 molL(-1). DGT validation experiments showed that Cr (VI) was measured accurately as well as selectively by PQAS DGT, whereas Cr (III) was not determined quantitatively. Compared with diphenylcarbazide spectrophotometric method (DPC), the measurement of Cr (VI) with PQAS DGT was agreement with that of DPC method in the industrial wastewater. PQAS-DGT device had been successfully deployed in local freshwater. The concentrations of Cr (VI) determined by PQAS DGT coupled with GFAAS in Nuer River, Ling River and North Lake were 0.73 ± 0.09 μg L(-1), 0.50 ± 0.07 μg L(-1) and 0.61 ± 0.07 μg L(-1), respectively. The results indicate that PQAS DGT device can be used for the selective sampling and measurement Cr (VI) in water and its detection limit is lower than that of DPC method. PMID:24632368

  7. Remodeling of host phosphatidylcholine by Chlamydia acyltransferase is regulated by acyl-CoA binding protein ACBD6 associated with lipid droplets

    PubMed Central

    Soupene, Eric; Wang, Derek; Kuypers, Frans A

    2015-01-01

    The bacterial human pathogen Chlamydia trachomatis invades cells as an infectious elementary body (EB). The EB is internalized into a vacuole that is hidden from the host defense mechanism, and is modified to sustain the development of the replicative reticulate body (RB). Inside this parasitophorous compartment, called the inclusion, the pathogen survives supported by an active exchange of nutrients and proteins with the host cell. We show that host lipids are scavenged and modified into bacterial-specific lipids by the action of a shared human-bacterial acylation mechanism. The bacterial acylating enzymes for the essential lipids 1-acyl-sn-glycerol 3-phosphate and 1-acyl-sn-phosphatidylcholine were identified as CT453 and CT775, respectively. Bacterial CT775 was found to be associated with lipid droplets (LDs). During the development of C. trachomatis, the human acyl-CoA carrier hACBD6 was recruited to cytosolic LDs and translocated into the inclusion. hACBD6 protein modulated the activity of CT775 in an acyl-CoA dependent fashion and sustained the activity of the bacterial acyltransferase by buffering the concentration of acyl-CoAs. We propose that disruption of the binding activity of the acyl-CoA carrier might represent a new drug-target to prevent growth of C. trachomatis. PMID:25604091

  8. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

    PubMed

    Leatherbarrow, R J; Rademacher, T W; Dwek, R A; Woof, J M; Clark, A; Burton, D R; Richardson, N; Feinstein, A

    1985-04-01

    Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events. PMID:4033665

  9. Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA*

    PubMed Central

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-01-01

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA. PMID:25542899

  10. Crystal structure of DNA cytidine deaminase ABOBEC3G catalytic deamination domain suggests a binding mode of full-length enzyme to single-stranded DNA.

    PubMed

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-02-13

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA. PMID:25542899

  11. Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.

    PubMed

    Scorsato, Valéria; Lima, Tatiani B; Righetto, Germanna L; Zanchin, Nilson I T; Brandão-Neto, José; Sandy, James; Pereira, Humberto D'Muniz; Ferrari, Állan J R; Gozzo, Fabio C; Smetana, Juliana H C; Aparicio, Ricardo

    2016-01-01

    TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase's active site, providing a structural framework for the function of TIPRL in PP2A inhibition. PMID:27489114

  12. Identification of a Binding Site for ASF/SF2 on an RNA Fragment Derived from the Hepatitis delta Virus Genome

    PubMed Central

    Sikora, Dorota; Zhang, Dajiang; Bojic, Teodora; Beeharry, Yasnee; Tanara, Ali; Pelchat, Martin

    2013-01-01

    The hepatitis delta virus (HDV) is a small (∼1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used. PMID:23349975

  13. The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions.

    PubMed

    Little, Richard; Dixon, Ray

    2003-08-01

    The expression of genes required for the synthesis of molybdenum nitrogenase in Azotobacter vinelandii is controlled by the NifL-NifA transcriptional regulatory complex in response to nitrogen, carbon, and redox status. Activation of nif gene expression by the transcriptional activator NifA is inhibited by direct protein-protein interaction with NifL under conditions unfavorable for nitrogen fixation. We have recently shown that the NifL-NifA system responds directly to physiological concentrations of 2-oxoglutarate, resulting in relief of NifA activity from inhibition by NifL under conditions when fixed nitrogen is limiting. The inhibitory activity of NifL is restored under conditions of excess combined nitrogen through the binding of the signal transduction protein Av GlnK to the carboxyl-terminal domain of NifL. The amino-terminal domain of NifA comprises a GAF domain implicated in the regulatory response to NifL. A truncated form of NifA lacking this domain is not responsive to 2-oxoglutarate in the presence of NifL, suggesting that the GAF domain is required for the response to this ligand. Using isothermal titration calorimetry, we demonstrate stoichiometric binding of 2-oxoglutarate to both the isolated GAF domain and the full-length A. vinelandii NifA protein with a dissociation constant of approximately 60 microm. Limited proteolysis experiments indicate that the binding of 2-oxoglutarate increases the susceptibility of the GAF domain to trypsin digestion and also prevents NifL from protecting these cleavage sites. However, protection by NifL is restored when the non-modified (non-uridylylated) form of Av GlnK is also present. Our results suggest that the binding of 2-oxoglutarate to the GAF domain of NifA may induce a conformational change that prevents inhibition by NifL under conditions when fixed nitrogen is limiting. PMID:12759352

  14. Structure of an Odorant-Vinding Protein form the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive

    SciTech Connect

    N Leite; R Krogh; W Xu; Y Ishida; J Iulek; W Leal; G Oliva

    2011-12-31

    The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 {angstrom} resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six {alpha}-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this 'lid' may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

  15. Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus

    PubMed Central

    Scorsato, Valéria; Lima, Tatiani B.; Righetto, Germanna L.; Zanchin, Nilson I. T.; Brandão-Neto, José; Sandy, James; Pereira, Humberto D’Muniz; Ferrari, Állan J. R.; Gozzo, Fabio C.; Smetana, Juliana H. C.; Aparicio, Ricardo

    2016-01-01

    TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition. PMID:27489114

  16. Novel β-Propeller of the BTB-Kelch Protein Krp1 Provides a Binding Site for Lasp-1 That Is Necessary for Pseudopodial Extension*♦

    PubMed Central

    Gray, Christopher H.; McGarry, Lynn C.; Spence, Heather J.; Riboldi-Tunnicliffe, Alan; Ozanne, Bradford W.

    2009-01-01

    Kelch-related protein 1 (Krp1) is up-regulated in oncogene-transformed fibroblasts. The Kelch repeats interact directly with the actin-binding protein Lasp-1 in membrane ruffles at the tips of pseudopodia, where both proteins are necessary for pseudopodial elongation. Herein, we investigate the molecular basis for this interaction. Probing an array of overlapping decapeptides of Rattus norvegicus (Rat) Krp1 with recombinant Lasp-1 revealed two binding sites; one (317YDPMENECYLT327) precedes the first of five Kelch repeats, and the other (563TEVNDIWKYEDD574) is in the last of the five Kelch repeats. Mutational analysis established that both binding sites are necessary for Krp1-Lasp-1 interaction in vitro and function in vivo. The crystal structure of the C-terminal domain of rat Krp1 (amino acids 289–606) reveals that both binding sites are brought into close proximity by the formation of a novel six-bladed β-propeller, where the first blade is not formed by a Kelch repeat. PMID:19726686

  17. African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT

    PubMed Central

    Miskin, James E.; Abrams, Charles C.; Dixon, Linda K.

    2000-01-01

    The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-κB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L. PMID:11000210

  18. Performance of the MM/GBSA scoring using a binding site hydrogen bond network-based frame selection: the protein kinase case.

    PubMed

    Adasme-Carreño, Francisco; Muñoz-Gutierrez, Camila; Caballero, Julio; Alzate-Morales, Jans H

    2014-07-21

    A conformational selection method, based on hydrogen bond (Hbond) network analysis, has been designed in order to rationalize the configurations sampled using molecular dynamics (MD), which are commonly used in the estimation of the relative binding free energy of ligands to macromolecules through the MM/GBSA or MM/PBSA method. This approach makes use of protein-ligand complexes obtained from X-ray crystallographic data, as well as from molecular docking calculations. The combination of several computational approaches, like long MD simulations on protein-ligand complexes, Hbond network-based selection by scripting techniques and finally MM/GBSA, provides better statistical correlations against experimental binding data than previous similar reported studies. This approach has been successfully applied in the ranking of several protein kinase inhibitors (CDK2, Aurora A and p38), which present both diverse and related chemical structures. PMID:24901037

  19. AUF-1 and YB-1 independently regulate β-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein

    PubMed Central

    van Zalen, Sebastiaan; Lombardi, Alyssa A.; Jeschke, Grace R.; Hexner, Elizabeth O.; Russell, J. Eric

    2015-01-01

    The normal expression of β-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of β-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP β-complex on the β-globin 3′UTR. Here, we demonstrate that the β-complex organizes during the erythropoietic interval when both β-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link β-complex assembly to the half-life of β-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both β-complex assembly and β-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the β-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the β-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of β-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1-and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs. PMID:25720531

  20. Alpha-DNA. IV: Alpha-anomeric and beta-anomeric tetrathymidylates covalently linked to intercalating oxazolopyridocarbazole. Synthesis, physicochemical properties and poly (rA) binding.

    PubMed Central

    Gautier, C; Morvan, F; Rayner, B; Huynh-Dinh, T; Igolen, J; Imbach, J L; Paoletti, C; Paoletti, J

    1987-01-01

    A new set of molecules made of an intercalating agent (oxazolopyridocarbazole, OPC) covalently linked through a polymethylene chain of various length to the 3' end of alpha-anomeric or beta-anomeric tetradeoxynucleotides (alpha- or beta-T4) have been synthesized. The beta-thymidylate modified compound (beta-T4C5OPC) is able to interact with the complementary sequence, beta-poly (rA); this interaction is strongly stabilized compared to the parent compound, beta-oligo(dT)4 and is specific for poly (rA). The molecule synthesized from the unnatural alpha-anomer, alpha-T4C5OPC, is also able to interact with poly (rA) leading to the formation of an alpha-beta hybrid stabilized by the energy provided by the OPC moiety. The stoechiometry of the binding reaction shows that an A-T pairing occurs in the alpha-beta heterohybrids. Tm studies reveal that the alpha-beta heterohybrids are more stable than their beta-beta counterparts. PMID:3628001

  1. Ly6 family member C4.4A binds laminins 1 and 5, associates with galectin-3 and supports cell migration.

    PubMed

    Paret, Claudia; Bourouba, Mehdi; Beer, Alexander; Miyazaki, Kaoru; Schnölzer, Martina; Fiedler, Sabine; Zöller, Margot

    2005-07-10

    C4.4A is a member of the Ly6 family, with low homology to uPAR. It has been detected mainly on metastasizing carcinoma cells and proposed to be involved in wound healing. So far, C4.4A has been observed as an orphan receptor, and its functional activity has not been explored. Using recombinant rat C4.4A (rrC4.4A) made in a eukaryotic expression system, we demonstrate by immunohistology that C4.4A ligands are strongly expressed in tissues adjacent to squamous epithelia of, e.g., tongue and esophagus, the expression pattern partly overlapping with laminin (LN) and complementing the C4.4A expression that is found predominantly on the basal layers of squamous epithelium. ELISA screening of several components of the extracellular matrix revealed selective binding of rrC4.4A to LN1 and LN5 and that transfection of the BSp73AS tumor line with C4.4A cDNA (BSp73AS-1B1) promoted LN1 and LN5 binding. Binding of BSp73AS-1B1 to LN5 and, less markedly, LN1 induced spreading, lamellipodia formation and migration. C4.4A also associates with galectin-3 in nontransformed tissues and tumor lines. There is evidence that the association of C4.4A with galectin-3 influences LN adhesion. C4.4A was described originally as a metastasis-associated molecule. Our findings that LN1 and LN5 are C4.4A ligands, that galectin-3 associates with C4.4A and that C4.4A ligand binding confers a migratory phenotype are well in line with the supposed metastasis association. PMID:15729693

  2. Phase 2 report on the evaluation of polyacrylonitrile (PAN) as a binding polymer for absorbers used to treat liquid radioactive wastes

    SciTech Connect

    Sebesta, F.; John, J.; Motl, A.

    1996-05-01

    The performance of PAN-based composite absorbers was evaluated in dynamic experiments at flow rates ranging from 25--100 bed volumes (BV) per hour. Composite absorbers with active components of ammonium molybdophosphate (AMP) PAN and K-Co ferrocyanide (KCoFC) PAN were used for separating Cs from a 1 M HNO{sub 3} + 1 M NaNO{sub 3} + 2 {times} 10{sup {minus}5} M CsCl acidic simulant solution. KCoFC-PAN and two other FC-based composite absorbers were tested for separating Cs from alkaline simulant solutions containing 0.01 M to 1 M NaOH and 1 M NaNO{sub 3} + x {times} 10{sup {minus}4} M CsCl. The efficiency of the Cs sorption on the AMP-PAN absorber from acidic simulant solutions was negatively influenced by the dissolution of the AMP active component. At flow rates of 50 BV/hr, the decontamination factor of about 10{sup 3} could be maintained for treatment of 380 BV of the feed. With the KCoFC-PAN absorber, the decontamination factor of about 10{sup 3} could be maintained for a feed volume as great as 1,800 BV. In alkaline simulant solutions, significant decomposition of the active components was observed, and the best performance was exhibited by the KCoFC-PAN absorber. Introductory experiments confirmed that Cs may be washed out of the composite absorbers. Regeneration of both absorbers for repetitive use was also found to be possible. The main result of the study is that PAN was proven to be a versatile polymer capable of forming porous composite absorbers with a large number of primary absorbers. The composite absorbers proved to be capable of withstanding the harsh acidic and alkaline conditions and significant radiation doses that may be expected in the treatment of US DOE wastes. A field demonstration is proposed as a follow-on activity.

  3. Activated RhoA Binds to the Pleckstrin Homology (PH) Domain of PDZ-RhoGEF, a Potential Site for Autoregulation

    SciTech Connect

    Chen, Zhe; Medina, Frank; Liu, Mu-ya; Thomas, Celestine; Sprang, Stephen R.; Sternweis, Paul C.

    2010-07-19

    Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH {center_dot} PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA {center_dot} GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH {center_dot} PH-RhoA {center_dot} GTP{gamma}S (guanosine 5{prime}-O-[{gamma}-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH {center_dot} PH do not overlap, and a ternary complex of PRG-DH {center_dot} PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.

  4. The Exosporium of B.cereus Contains a Binding Site for gC1qR/p33: Implication in Spore Attachment and/or Entry.

    SciTech Connect

    GHEBREHIWET,B.; TANTRAL, L.; TITMUS, M.A.; PANESSA-WARREN, B.J.; TORTORA, G.T.; WONG, S.S.; WARREN, J.B.

    2008-01-01

    B. cereus, is a member of a genus of aerobic, gram-positive, spore-forming rod-like bacilli, which includes the deadly, B. anthracis. Preliminary experiments have shown that gC1qR binds to B.cereus spores that have been attached to microtiter plates. The present studies were therefore undertaken, to examine if cell surface gC1qR plays a role in B.cereus spore attachment and/or entry. Monolayers of human colon carcinoma (Caco-2) and lung cells were grown to confluency on 6 mm coverslips in shell vials with gentle swirling in a shaker incubator. Then, 2 {micro}l of a suspension of strain SB460 B.cereus spores (3x10{sup 8}/ml, in sterile water), were added and incubated (1-4 h; 36{sup 0} C) in the presence or absence of anti-gC1qR mAb-carbon nanoloops. Examination of these cells by EM revealed that: (1) When B. cereus endospores contacted the apical Caco-2 cell surface, or lung cells, gClqR was simultaneously detectable, indicating upregulation of the molecule. (2) In areas showing spore contact with the cell surface, gClqR expression was often adjacent to the spores in association with microvilli (Caco-2 cells) or cytoskeletal projections (lung cells). (3) Furthermore, the exosporia of the activated and germinating spores were often decorated with mAb-nanoloops. These observations were further corroborated by experiments in which B.cereus spores were readily taken up by monocytes and neutrophils, and this uptake was partially inhibited by mAb 60.11, which recognizes the C1q binding site on gC1qR. Taken together, the data suggest a role, for gC1qR at least in the initial stages of spore attachment and/or entry.

  5. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  6. Insulin-like Growth Factor-II (IGF-II) and IGF-II Analogs with Enhanced Insulin Receptor-a Binding Affinity Promote Neural Stem Cell Expansion*

    PubMed Central

    Ziegler, Amber N.; Chidambaram, Shravanthi; Forbes, Briony E.; Wood, Teresa L.; Levison, Steven W.

    2014-01-01

    The objective of this study was to employ genetically engineered IGF-II analogs to establish which receptor(s) mediate the stemness promoting actions of IGF-II on mouse subventricular zone neural precursors. Neural precursors from the subventricular zone were propagated in vitro in culture medium supplemented with IGF-II analogs. Cell growth and identity were analyzed using sphere generation and further analyzed by flow cytometry. F19A, an analog of IGF-II that does not bind the IGF-2R, stimulated an increase in the proportion of neural stem cells (NSCs) while decreasing the proportion of the later stage progenitors at a lower concentration than IGF-II. V43M, which binds to the IGF-2R with high affinity but which has low binding affinity to the IGF-1R and to the A isoform of the insulin receptor (IR-A) failed to promote NSC growth. The positive effects of F19A on NSC growth were unaltered by the addition of a functional blocking antibody to the IGF-1R. Altogether, these data lead to the conclusion that IGF-II promotes stemness of NSCs via the IR-A and not through activation of either the IGF-1R or the IGF-2R. PMID:24398690

  7. Sodium polyacrylate as a binding agent in diffusive gradients in thin-films technique for the measurement of Cu2+ and Cd2+ in waters.

    PubMed

    Fan, Hongtao; Sun, Ting; Li, Weijia; Sui, Dianpeng; Jin, Shuang; Lian, Xiaojing

    2009-10-15

    An aqueous solution containing sodium polyacrylate (PA, 0.0030M) was used in diffusive gradients in thin-films technique (DGT) to measure DGT-labile Cu(2+) and Cd(2+) concentrations. The DGT devices (PA DGT) were validated in four types of solutions, including synthetic river waters containing metal ions with or without complexing EDTA, natural river water (Hun River, Shenyang, China) spiked with Cu(2+) and Cd(2+), and an industrial wastewater (Shenyang, China). Results showed that only free metal ions were measured by PA DGT, recovery=98.79% for Cu(2+) and recovery=97.80% for Cd(2+) in solutions containing only free metal ions, recovery=51.02% for Cu(2+) and recovery=51.92% for Cd(2+) in solution with metal/EDTA molar ratio of 2:1 and recovery=0 in solutions with metal/EDTA molar ratio of 1:1 and 1:2. These indicated that the complexes of Cu-EDTA and Cd-EDTA were DGT-inert or not DGT-labile. The DGT performance in spiked river water (recovery=8.47% for Cu(2+) and recovery=27.48% for Cd(2+)) and in industrial wastewater (recovery=14.16% for Cd(2+)) were also investigated. Conditional stability constants (logK) of PA-Cu and PA-Cd complexes were determined as 6.98 and 5.61, respectively, indicating strong interaction between PA and the metals. PMID:19635351

  8. Conformation-Specific Infrared and Ultraviolet Spectroscopy of DIBENZO-15-CROWN-5-(H2O)1-CLUSTER: Reshaping a Binding Pocket

    NASA Astrophysics Data System (ADS)

    Buchanan, Evan G.; Rodrigo, Chirantha P.; Gutberlet, Anna K.; Zwier, Timothy S.

    2010-06-01

    Crown ethers are oxygen containing macrocycles noted for their ability to preferentially bind substrates such as ions and water. Despite the high symmetry inherent to the chemical structure, crown ethers are remarkably flexible, adapting their conformation to the substrate to which they are bound. Here, we present the conformational preferences of the singly hydrated dibenzo-15-crown-5 ether (DB15C) complex formed and cooled in a supersonic jet. The resonance enhanced two-photon ionization, UV-UV Hole-burning, and resonant ion-dip infrared spectra lead to the identification of a single DB15C-(H2O)1 conformer with the water doubly hydrogen bonded to the crown. Single vibronic level dispersed fluorescence identified both electronic origins and the coupling between the two chromophores. Finally, infrared population transfer spectroscopy is used to study the monomer conformer populations formed by infrared photodissocation of the complex via the water OH stretch transitions, providing unique insight to the energy flow between water and crown.

  9. Carbonation as a binding mechanism for coal/calcium hydroxide pellets. Final technical report, 1 September, 1992--31 August, 1993

    SciTech Connect

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

    1993-12-31

    This research was 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, was studied as a method for improving pellet quality. Carbonation forms a cementitious matrix of calcium carbonate. Research has demonstrated that calcium hydroxide is a viable binder for coal fines and that a roller-and-die pellet mill is an effective method of pellet formation. From a minus 28 mesh preparation plant fine coal sample, a roller-and-die pellet mill produced strong pellets when 5 and 10% calcium hydroxide was used as a binder. The pellets containing 10% calcium hydroxide strengthened considerably when air cured. This increase in strength was attributed to carbonation via atmospheric carbon dioxide. Pellets containing 10 wt% calcium hydroxide were produced using an extruder but pellets formed in this manner were much weaker than pellets produced with the roller-and-die mill. In tests performed using a laboratory hydraulic press, the effect of particle size and compaction pressure on pellet strength was studied. 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. As above, this increase in strength was attributed to carbonation of the calcium hydroxide via atmospheric carbon dioxide.

  10. The C-Terminus of Human Copper Importer Ctr1 Acts as a Binding Site and Transfers Copper to Atox1.

    PubMed

    Kahra, Dana; Kovermann, Michael; Wittung-Stafshede, Pernilla

    2016-01-01

    Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ∼10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones. PMID:26745413

  11. Mutation of Arg-115 of human class III alcohol dehydrogenase: a binding site required for formaldehyde dehydrogenase activity and fatty acid activation.

    PubMed Central

    Engeland, K; Höög, J O; Holmquist, B; Estonius, M; Jörnvall, H; Vallee, B L

    1993-01-01

    The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escherichia coli and purified by affinity chromatography and ion-exchange HPLC. The activities of the recombinant native and mutant enzymes toward ethanol are essentially identical, but mutagenesis greatly decreases the kcat/Km values for glutathione-dependent formaldehyde oxidation. The catalytic efficiency for the Asp variant is < 0.1% that of the unmutated enzyme, due to both a higher Km and a lower kcat value. As with the native enzyme, neither mutant can oxidize methanol, be saturated by ethanol, or be inhibited by 4-methylpyrazole; i.e., they retain these class III characteristics. In contrast, however, their activation by fatty acids, another characteristic unique to class III alcohol dehydrogenase, is markedly attenuated. The Ala mutant is activated only slightly, but the Asp mutant is not activated at all. The results strongly indicate that Arg-115 in class III alcohol dehydrogenase is a component of the binding site for activating fatty acids and is critical for the binding of S-hydroxymethylglutathione in glutathione-dependent formaldehyde dehydrogenase activity. PMID:8460164

  12. Pithy Protection: Nicotiana attenuata’s Jasmonic Acid-Mediated Defenses Are Required to Resist Stem-Boring Weevil Larvae1[W][OA

    PubMed Central

    Diezel, Celia; Kessler, Danny; Baldwin, Ian T.

    2011-01-01

    Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C6 aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant’s apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata’s fitness. PMID:21300916

  13. Burkholderia cenocepacia ShvR-regulated genes that influence colony morphology, biofilm formation, and virulence.

    PubMed

    Subramoni, Sujatha; Nguyen, David T; Sokol, Pamela A

    2011-08-01

    Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia. PMID:21690240

  14. A liver-specific defect of Acyl-CoA degradation produces hyperammonemia, hypoglycemia and a distinct hepatic Acyl-CoA pattern.

    PubMed

    Gauthier, Nicolas; Wu, Jiang Wei; Wang, Shu Pei; Allard, Pierre; Mamer, Orval A; Sweetman, Lawrence; Moser, Ann B; Kratz, Lisa; Alvarez, Fernando; Robitaille, Yves; Lépine, François; Mitchell, Grant A

    2013-01-01

    Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-(14)C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication. PMID:23861731

  15. Unexpected functional diversity in the fatty acid desaturases of the flour beetle Tribolium castaneum and identification of key residues determining activity.

    PubMed

    Haritos, Victoria S; Horne, Irene; Damcevski, Katherine; Glover, Karen; Gibb, Nerida

    2014-08-01

    Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity. PMID:24880119

  16. Acetyl-L-carnitine and lipoic acid improve mitochondrial abnormalities and serum levels of liver enzymes in a mouse model of nonalcoholic fatty liver disease.

    PubMed

    Kathirvel, Elango; Morgan, Kengathevy; French, Samuel W; Morgan, Timothy R

    2013-11-01

    Mitochondrial abnormalities are suggested to be associated with the development of nonalcoholic fatty liver. Liver mitochondrial content and function have been shown to improve in oral feeding of acetyl-L-carnitine (ALC) to rodents. Carnitine is involved in the transport of acyl-coenzyme A across the mitochondrial membrane to be used in mitochondrial β-oxidation. We hypothesized that oral administration ALC with the antioxidant lipoic acid (ALC + LA) would benefit nonalcoholic fatty liver. To test our hypothesis, we fed Balb/C mice a standard diet (SF) or SF with ALC + LA or high-fat diet (HF) or HF with ALC + LA for 6 months. Acetyl-L-carnitine and LA were dissolved at 0.2:0.1% (wt/vol) in drinking water, and mice were allowed free access to food and water. Along with physical parameters, insulin resistance (blood glucose, insulin, glucose tolerance), liver function (alanine transaminase [ALT], aspartate transaminase [AST]), liver histology (hematoxylin and eosin), oxidative stress (malondialdehyde), and mitochondrial abnormalities (carbamoyl phosphate synthase 1 and electron microscopy) were done. Compared with SF, HF had higher body, liver, liver-to-body weight ratio, white adipose tissue, ALT, AST, liver fat, oxidative stress, and insulin resistance. Coadministration of ALC + LA to HF animals significantly improved the mitochondrial marker carbamoyl phosphate synthase 1 and the size of the mitochondria in liver. Alanine transaminase and AST levels were decreased. In a nonalcoholic fatty liver mice model, ALC + LA combination improved liver mitochondrial content, size, serum ALT, and AST without significant changes in oxidative stress, insulin resistance, and liver fat accumulation. PMID:24176233

  17. Lipidomic and Spatio-Temporal Imaging of Fat by Mass Spectrometry in Mice Duodenum during Lipid Digestion

    PubMed Central

    Seyer, Alexandre; Cantiello, Michela; Bertrand-Michel, Justine; Roques, Véronique; Nauze, Michel; Bézirard, Valérie; Touboul, David; Coméra, Christine

    2013-01-01

    Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy. PMID:23560035

  18. Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis

    PubMed Central

    LaMarca, B. Babbette D.; Zhu, Wenming; Arceneaux, Jean E. L.; Rowe Byers, B.; Lundrigan, Michael D.

    2004-01-01

    Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 μM Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an Rf of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an Rf of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin. PMID:14702306

  19. Reveal genes functionally associated with ACADS by a network study.

    PubMed

    Chen, Yulong; Su, Zhiguang

    2015-09-15

    Establishing a systematic network is aimed at finding essential human gene-gene/gene-disease pathway by means of network inter-connecting patterns and functional annotation analysis. In the present study, we have analyzed functional gene interactions of short-chain acyl-coenzyme A dehydrogenase gene (ACADS). ACADS plays a vital role in free fatty acid β-oxidation and regulates energy homeostasis. Modules of highly inter-connected genes in disease-specific ACADS network are derived by integrating gene function and protein interaction data. Among the 8 genes in ACADS web retrieved from both STRING and GeneMANIA, ACADS is effectively conjoined with 4 genes including HAHDA, HADHB, ECHS1 and ACAT1. The functional analysis is done via ontological briefing and candidate disease identification. We observed that the highly efficient-interlinked genes connected with ACADS are HAHDA, HADHB, ECHS1 and ACAT1. Interestingly, the ontological aspect of genes in the ACADS network reveals that ACADS, HAHDA and HADHB play equally vital roles in fatty acid metabolism. The gene ACAT1 together with ACADS indulges in ketone metabolism. Our computational gene web analysis also predicts potential candidate disease recognition, thus indicating the involvement of ACADS, HAHDA, HADHB, ECHS1 and ACAT1 not only with lipid metabolism but also with infant death syndrome, skeletal myopathy, acute hepatic encephalopathy, Reye-like syndrome, episodic ketosis, and metabolic acidosis. The current study presents a comprehensible layout of ACADS network, its functional strategies and candidate disease approach associated with ACADS network. PMID:26045367

  20. Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice

    PubMed Central

    Terasaki, Michishige; Hiromura, Munenori; Mori, Yusaku; Kohashi, Kyoko; Nagashima, Masaharu; Kushima, Hideki; Watanabe, Takuya; Hirano, Tsutomu

    2015-01-01

    Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe−/−) mice, streptozotocin-induced diabetic Apoe−/− mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe−/− mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe−/− mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe−/− mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo. PMID:26606676

  1. Changes in esculeoside A content in different regions of the tomato fruit during maturation and heat processing.

    PubMed

    Katsumata, Akiko; Kimura, Mizuki; Saigo, Hiromi; Aburaya, Kei; Nakano, Masako; Ikeda, Tsuyoshi; Fujiwara, Yukio; Nagai, Ryoji

    2011-04-27

    We previously demonstrated that esculeogenin A, a new aglycone of the tomato sapogenol esculeoside A, inhibits both acyl coenzyme A:cholesterol acyl-transferase (ACAT)-1 and -2 and ameliorates the pathogenesis of atherosclerosis in apoE deficient mice. Although we believe that daily intake of esculeoside A from tomato products can play a beneficial role in preventing the pathogenesis of atherosclerosis, the compound is not being used for preventive medicine due to the lack of information on methods for quantitative analysis and the content and stability of the compound in tomato products. In the present study, we report the development of a high-performance liquid chromatography (HPLC) method using an instrument equipped with a refractive index (RI) detector for esculeoside A quantification. We used this method to measure the changes in esculeoside A content during maturation, its distribution in the fruit body, and its stability during the heating process. The contents of esculeoside A in cherry tomatoes and Momotaro tomatoes were 21- and 9-fold, respectively, higher than that of lycopene, which is the most well-known compound in tomatoes. Furthermore, the esculeoside A content in pericarp wall was higher than in the whole tomato fruit and increased in a time-dependent manner during maturation. Although the melting point of purified esculeoside A was 225 °C, the esculeoside A in crude tomato extract decreased in a temperature-dependent manner. Degradation due to the heating process was inhibited under a pH of 9. These results demonstrated that the esculeoside A content differs in the various types of tomatoes, during maturation, and during the heating process used for preservation. PMID:21395308

  2. Effects of clofibrate treatment in laying hens.

    PubMed

    König, B; Kluge, H; Haase, K; Brandsch, C; Stangl, G I; Eder, K

    2007-06-01

    Expression of peroxisome proliferator-activated receptor-alpha (PPARalpha) has been shown in liver of chicks, but effects of its activation have not yet been investigated. In this study, laying hens were treated with clofibrate, a synthetic PPARalpha agonist, to investigate the effects of PPARalpha activation on liver lipid metabolism. Hens receiving a diet containing 5 g of clofibrate/kg had a lower food intake and higher liver mRNA concentrations of typical PPARalpha target genes (carnitine palmitoyltransferase 1A, acyl-coenzyme A oxidase, bifunctional enzyme, lipoprotein lipase) involved in hepatic mitochondrial and peroxisomal beta-oxidation and plasma triglyceride clearance than control hens that received the same diet without clofibrate (P<0.05). Hens treated with clofibrate also had lower mRNA concentrations of fatty acid synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and low-density lipoprotein receptor, proteins involved in fatty acid biosynthesis and cholesterol biosynthesis and uptake, than hens fed the control diet (P<0.05). These changes in clofibrate-treated hens were accompanied by reduced liver triglyceride concentrations, strongly diminished very low density triglyceride and cholesterol concentrations (P<0.05), a disturbed maturation of egg follicles, a complete stop of egg production, and a markedly reduced plasma 17-beta-estradiol concentration (P<0.05). In conclusion, it is shown that clofibrate has complex effects on hepatic lipid metabolism in laying hens that mimic PPARalpha activation in mammals, affect maturation of egg follicles, and lead to a stop of egg production. Because clofibrate treatment strongly reduced food intake in the hens, some of these effects (i.e., egg production) may have been due to a low energy and nutrient intake. PMID:17495091

  3. Intestinal Long-Chain Fatty Acids Act as a Direct Signal To Modulate Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System

    PubMed Central

    Ellermeier, Jeremy R.; Cott Chubiz, Jessica E.

    2016-01-01

    ABSTRACT Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the T3SS structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. Expression of the SPI1 genes is tightly controlled by numerous regulatory inputs to ensure proper timing in production of the T3SS apparatus. Loss of FadD, an acyl coenzyme A (acyl-CoA) synthetase required for degradation of long-chain fatty acids (LCFAs), was known to decrease hilA expression. We show that free external LCFAs repress expression of hilA independently of FadD and the LCFA degradation pathway. Genetic and biochemical evidence suggests that LCFAs act directly to block primarily HilD activity. Further analyses show that in the absence of FadD, hilA expression is downregulated due to endogenous production of free LCFAs, which are excreted into the culture medium via TolC and then transported back into the bacterial cell via FadL. A fadL mutant is more virulent than the wild-type strain in mouse oral competition assays independently of LCFA degradation, showing that, in the host, dietary LCFAs serve as a signal for proper regulation of SPI1 expression, rather than an energy source. PMID:26884427

  4. A dynamic, mechanistic model of metabolism in adipose tissue of lactating dairy cattle.

    PubMed

    McNamara, J P; Huber, K; Kenéz, A

    2016-07-01

    Research in dairy cattle biology has resulted in a large body of knowledge on nutrition and metabolism in support of milk production and efficiency. This quantitative knowledge has been compiled in several model systems to balance and evaluate rations and predict requirements. There are also systems models for metabolism and reproduction in the cow that can be used to support research programs. Adipose tissue plays a significant role in the success and efficiency of lactation, and recent research has resulted in several data sets on genomic differences and changes in gene transcription of adipose tissue in dairy cattle. To fully use this knowledge, we need to build and expand mechanistic, dynamic models that integrate control of metabolism and production. Therefore, we constructed a second-generation dynamic, mechanistic model of adipose tissue metabolism of dairy cattle. The model describes the biochemical interconversions of glucose, acetate, β-hydroxybutyrate (BHB), glycerol, C16 fatty acids, and triacylglycerols. Data gathered from our own research and published references were used to set equation forms and parameter values. Acetate, glucose, BHB, and fatty acids are taken up from blood. The fatty acids are activated to the acyl coenzyme A moieties. Enzymatically catalyzed reactions are explicitly described with parameters including maximal velocity and substrate sensitivity. The control of enzyme activity is partially carried out by insulin and norepinephrine, portraying control in the cow. Model behavior was adequate, with sensitive responses to changing substrates and hormones. Increased nutrient uptake and increased insulin stimulate triacylglycerol synthesis, whereas a reduction in nutrient availability or increase in norepinephrine increases triacylglycerol hydrolysis and free fatty acid release to blood. This model can form a basis for more sophisticated integration of existing knowledge and future studies on metabolic efficiency of dairy cattle. PMID

  5. The COMATOSE ATP-Binding Cassette Transporter Is Required for Full Fertility in Arabidopsis1[W][OA

    PubMed Central

    Footitt, Steven; Dietrich, Daniela; Fait, Aaron; Fernie, Alisdair R.; Holdsworth, Michael J.; Baker, Alison; Theodoulou, Frederica L.

    2007-01-01

    COMATOSE (CTS) encodes a peroxisomal ATP-binding cassette transporter required not only for β-oxidation of storage lipids during germination and establishment, but also for biosynthesis of jasmonic acid and conversion of indole butyric acid to indole acetic acid. cts mutants exhibited reduced fertilization, which was rescued by genetic complementation, but not by exogenous application of jasmonic acid or indole acetic acid. Reduced fertilization was also observed in thiolase (kat2-1) and peroxisomal acyl-Coenzyme A synthetase mutants (lacs6-1,lacs7-1), indicating a general role for β-oxidation in fertility. Genetic analysis revealed reduced male transmission of cts alleles and both cts pollen germination and tube growth in vitro were impaired in the absence of an exogenous carbon source. Aniline blue staining of pollinated pistils demonstrated that pollen tube growth was affected only when both parents bore the cts mutation, indicating that expression of CTS in either male or female tissues was sufficient to support pollen tube growth in vivo. Accordingly, abundant peroxisomes were detected in a range of maternal tissues. Although γ-aminobutyric acid levels were reduced in flowers of cts mutants, they were unchanged in kat2-1, suggesting that alterations in γ-aminobutyric acid catabolism do not contribute to the reduced fertility phenotype through altered pollen tube targeting. Taken together, our data support an important role for β-oxidation in fertility in Arabidopsis (Arabidopsis thaliana) and suggest that this pathway could play a role in the mobilization of lipids in both pollen and female tissues. PMID:17468211

  6. Loss of Apoptosis Regulator through Modulating IAP Expression (ARIA) Protects Blood Vessels from Atherosclerosis*

    PubMed Central

    Matsuo, Kiyonari; Akakabe, Yoshiki; Kitamura, Youhei; Shimoda, Yoshiaki; Ono, Kazunori; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Hatakeyama, Kinta; Asada, Yujiro; Emoto, Noriaki; Ikeda, Koji

    2015-01-01

    Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases. PMID:25533470

  7. A Liver-Specific Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern

    PubMed Central

    Gauthier, Nicolas; Wu, Jiang Wei; Wang, Shu Pei; Allard, Pierre; Mamer, Orval A.; Sweetman, Lawrence; Moser, Ann B.; Kratz, Lisa; Alvarez, Fernando; Robitaille, Yves; Lépine, François; Mitchell, Grant A.

    2013-01-01

    Most conditions detected by expanded newborn screening result from deficiency of one of the enzymes that degrade acyl-coenzyme A (CoA) esters in mitochondria. The role of acyl-CoAs in the pathophysiology of these disorders is poorly understood, in part because CoA esters are intracellular and samples are not generally available from human patients. We created a mouse model of one such condition, deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (HL), in liver (HLLKO mice). HL catalyses a reaction of ketone body synthesis and of leucine degradation. Chronic HL deficiency and acute crises each produced distinct abnormal liver acyl-CoA patterns, which would not be predictable from levels of urine organic acids and plasma acylcarnitines. In HLLKO hepatocytes, ketogenesis was undetectable. Carboxylation of [2-14C] pyruvate diminished following incubation of HLLKO hepatocytes with the leucine metabolite 2-ketoisocaproate (KIC). HLLKO mice also had suppression of the normal hyperglycemic response to a systemic pyruvate load, a measure of gluconeogenesis. Hyperammonemia and hypoglycemia, cardinal features of many inborn errors of acyl-CoA metabolism, occurred spontaneously in some HLLKO mice and were inducible by administering KIC. KIC loading also increased levels of several leucine-related acyl-CoAs and reduced acetyl-CoA levels. Ultrastructurally, hepatocyte mitochondria of KIC-treated HLLKO mice show marked swelling. KIC-induced hyperammonemia improved following administration of carglumate (N-carbamyl-L-glutamic acid), which substitutes for the product of an acetyl-CoA-dependent reaction essential for urea cycle function, demonstrating an acyl-CoA-related mechanism for this complication. PMID:23861731

  8. Overexpression of a BAHD acyltransferase, OsAt10, alters rice cell wall hydroxycinnamic acid content and saccharification.

    PubMed

    Bartley, Laura E; Peck, Matthew L; Kim, Sung-Ryul; Ebert, Berit; Manisseri, Chithra; Chiniquy, Dawn M; Sykes, Robert; Gao, Lingfang; Rautengarten, Carsten; Vega-Sánchez, Miguel E; Benke, Peter I; Canlas, Patrick E; Cao, Peijian; Brewer, Susan; Lin, Fan; Smith, Whitney L; Zhang, Xiaohan; Keasling, Jay D; Jentoff, Rolf E; Foster, Steven B; Zhou, Jizhong; Ziebell, Angela; An, Gynheung; Scheller, Henrik V; Ronald, Pamela C

    2013-04-01

    Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed. PMID:23391577

  9. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  10. Microsomal Lyso-Phosphatidic Acid Acyltransferase from a Brassica oleracea Cultivar Incorporates Erucic Acid into the sn-2 Position of Seed Triacylglycerols.

    PubMed Central

    Taylor, D. C.; Barton, D. L.; Giblin, E. M.; MacKenzie, S. L.; Van Den Berg, CGJ.; McVetty, PBE.

    1995-01-01

    Developing seeds from Brassica oleracea (L.) var botrytis cv Sesam were examined for the ability to biosynthesize and incorporate erucic acid into triacylglycerols (TAGs). Seed embryos at mid-development contained a high concentration of erucic acid in diacylglycerols and TAGs, and substantial levels were also detected in free fatty acids, acyl-coenzyme A (CoA), phosphatidic acid, and phosphatidylcholine. Homogenates and microsomal fractions from seeds at mid-development produced [14C]eicosenoyl- and [14C]erucoyl-CoAs from [14C]oleoyl-CoA in the presence of malonyl-CoA and reducing equivalents in vitro. These fatty acids were incorporated into TAGs via the Kennedy pathway. However, unlike most Brassicaceae, the B. oleracea was able to insert significant erucic acid into the sn-2 position of TAGs. It was shown that the lyso-phosphatidic acid acyltransferase (LPAT) incorporated erucic acid into the sn-2 position of lyso-phosphatidic acid. The erucoyl-CoA:LPAT activity during seed development and the sn-2 erucic acid content of the TAG fraction in mature seed were compared to those in B. napus, Tropaeolum majus, and Limnanthes douglasii. There was a correlation between the in vitro erucoyl-CoA:LPAT activity and the sn-2 erucic acid content in seed TAGs. To our knowledge, this is the first member of the Brassicaceae reported to have an LPAT able to use erucoyl-CoA. This observation has important implications for efforts being made to increase the erucic acid content in B. napus, to supply strategic industrial feedstocks. PMID:12228602

  11. Bardoxolone methyl prevents insulin resistance and the development of hepatic steatosis in mice fed a high-fat diet.

    PubMed

    Camer, Danielle; Yu, Yinghua; Szabo, Alexander; Dinh, Chi H L; Wang, Hongqin; Cheng, Licai; Huang, Xu-Feng

    2015-09-01

    High-fat (HF) diet-induced obesity is a major risk factor for the development of insulin resistance and hepatic steatosis. We examined the hypothesis that bardoxolone methyl (BM) would prevent the development of insulin resistance and hepatic steatosis in mice fed a HF diet. C57BL/6J male mice were fed a lab chow (LC), HF (40% fat), or HF diet supplemented with 10 mg/kg/day BM orally for 21 weeks. Glucose metabolism was assessed using a glucose tolerance test (GTT) and insulin sensitivity test (IST). Signalling molecules involved in insulin resistance, inflammation, and lipid metabolism were examined in liver tissue via western blotting and RT-PCR. BM prevented HF diet-induced insulin resistance and alterations in the protein levels of protein tyrosine phosphatase 1B (PTP1B), forkhead box protein O1 (FOXO1) and BDNF, and expression of the insulin receptor (IR), IRS-1 and glucose-6-phosphatase (G6Pase) genes. Furthermore, BM prevented fat accumulation in the liver and decreases in the β-oxidation gene, peroxisomal acyl-coenzyme A oxidase 1 (ACOX) in mice fed a HF diet. In the livers of HF fed mice, BM administration prevented HF diet-induced macrophage infiltration, inflammation as indicated by reduced IL-6 and signal transducer and activator of transcription 3 (STAT3) protein levels and TNFα mRNA expression, and increased nuclear factor-like 2 (Nrf2) mRNA expression and nuclear protein levels. These findings suggest that BM prevents HF diet induced insulin resistance and the development of hepatic steatosis in mice fed a chronic HF diet through modulation of molecules involved in insulin signalling, lipid metabolism and inflammation in the liver. PMID:26001833

  12. A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

    PubMed Central

    Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

    2012-01-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  13. Genetic Examination of Initial Amino Acid Oxidation and Glutamate Catabolism in the Hyperthermophilic Archaeon Thermococcus kodakarensis

    PubMed Central

    Yokooji, Yuusuke; Sato, Takaaki; Fujiwara, Shinsuke; Imanaka, Tadayuki

    2013-01-01

    Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (ΔpyrF ΔtrpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP+/NAD+) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis. PMID:23435976

  14. A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution.

    PubMed

    Yang, Weili; Simpson, Jeffrey P; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B

    2012-10-01

    Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

  15. Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coli▿†

    PubMed Central

    Lennen, Rebecca M.; Kruziki, Max A.; Kumar, Kritika; Zinkel, Robert A.; Burnum, Kristin E.; Lipton, Mary S.; Hoover, Spencer W.; Ranatunga, Don R.; Wittkopp, Tyler M.; Marner, Wesley D.; Pfleger, Brian F.

    2011-01-01

    Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, long-chain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli. PMID:21948837

  16. Loss of apoptosis regulator through modulating IAP expression (ARIA) protects blood vessels from atherosclerosis.

    PubMed

    Matsuo, Kiyonari; Akakabe, Yoshiki; Kitamura, Youhei; Shimoda, Yoshiaki; Ono, Kazunori; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Hatakeyama, Kinta; Asada, Yujiro; Emoto, Noriaki; Ikeda, Koji

    2015-02-01

    Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases. PMID:25533470

  17. Amelioration of Hyperglycemia with a Sodium-Glucose Cotransporter 2 Inhibitor Prevents Macrophage-Driven Atherosclerosis through Macrophage Foam Cell Formation Suppression in Type 1 and Type 2 Diabetic Mice.

    PubMed

    Terasaki, Michishige; Hiromura, Munenori; Mori, Yusaku; Kohashi, Kyoko; Nagashima, Masaharu; Kushima, Hideki; Watanabe, Takuya; Hirano, Tsutomu

    2015-01-01

    Direct associations between hyperglycemia and atherosclerosis remain unclear. We investigated the association between the amelioration of glycemia by sodium-glucose cotransporter 2 inhibitors (SGLT2is) and macrophage-driven atherosclerosis in diabetic mice. We administered dapagliflozin or ipragliflozin (1.0 mg/kg/day) for 4-weeks to apolipoprotein E-null (Apoe-/-) mice, streptozotocin-induced diabetic Apoe-/- mice, and diabetic db/db mice. We then determined aortic atherosclerosis, oxidized low-density lipoprotein (LDL)-induced foam cell formation, and related gene expression in exudate peritoneal macrophages. Dapagliflozin substantially decreased glycated hemoglobin (HbA1c) and glucose tolerance without affecting body weight, blood pressure, plasma insulin, and lipids in diabetic Apoe-/- mice. Aortic atherosclerotic lesions, atheromatous plaque size, and macrophage infiltration in the aortic root increased in diabetic Apoe-/- mice; dapagliflozin attenuated these changes by 33%, 27%, and 20%, respectively. Atherosclerotic lesions or foam cell formation highly correlated with HbA1c. Dapagliflozin did not affect atherosclerosis or plasma parameters in non-diabetic Apoe-/- mice. In db/db mice, foam cell formation increased by 4-fold compared with C57/BL6 mice, whereas ipragliflozin decreased it by 31%. Foam cell formation exhibited a strong correlation with HbA1c. Gene expression of lectin-like ox-LDL receptor-1 and acyl-coenzyme A:cholesterol acyltransferase 1 was upregulated, whereas that of ATP-binding cassette transporter A1 was downregulated in the peritoneal macrophages of both types of diabetic mice. SGLT2i normalized these gene expressions. Our study is the first to demonstrate that SGLT2i exerts anti-atherogenic effects by pure glucose lowering independent of insulin action in diabetic mice through suppressing macrophage foam cell formation, suggesting that foam cell formation is highly sensitive to glycemia ex vivo. PMID:26606676

  18. Sex Differences in Global mRNA Content of Human Skeletal Muscle

    PubMed Central

    Maher, Amy C.; Fu, Minghua H.; Isfort, Robert J.; Varbanov, Alex R.; Qu, Xiaoyan A.; Tarnopolsky, Mark A.

    2009-01-01

    Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, β-hydroxyacyl-CoA dehydrogenase, CD36). One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2), trifunctional protein β (HADHB), catalase, lipoprotein lipase (LPL), and uncoupling protein-2 (UCP-2) were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI), peroxisome proliferator-activated receptor (PPAR)δ were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARδ, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally “primed” for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined. PMID:19623254

  19. Analysis of lipid profile in lipid storage myopathy.

    PubMed

    Aguennouz, M'hammed; Beccaria, Marco; Purcaro, Giorgia; Oteri, Marianna; Micalizzi, Giuseppe; Musumesci, Olimpia; Ciranni, Annmaria; Di Giorgio, Rosa Maria; Toscano, Antonio; Dugo, Paola; Mondello, Luigi

    2016-09-01

    Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13-24% range, C18:1n9 in the 20-52% range, and C18:2n6 in the 10-25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct

  20. Three TaFAR genes function in the biosynthesis of primary alcohols and the response to abiotic stresses in Triticum aestivum.

    PubMed

    Wang, Meiling; Wang, Yong; Wu, Hongqi; Xu, Jing; Li, Tingting; Hegebarth, Daniela; Jetter, Reinhard; Chen, Letian; Wang, Zhonghua

    2016-01-01

    Cuticular waxes play crucial roles in protecting plants against biotic and abiotic stresses. They are complex mixtures of very-long-chain fatty acids and their derivatives, including C20-C32 fatty alcohols. Here, we report the identification of 32 FAR-like genes and the detailed characterization of TaFAR2, TaFAR3 and TaFAR4, wax biosynthetic genes encoding fatty acyl-coenzyme A reductase (FAR) in wheat leaf cuticle. Heterologous expression of the three TaFARs in wild-type yeast and mutated yeast showed that TaFAR2, TaFAR3 and TaFAR4 were predominantly responsible for the accumulation of C18:0, C28:0 and C24:0 primary alcohols, respectively. Transgenic expression of the three TaFARs in tomato fruit and Arabidopsis cer4 mutant led to increased production of C22:0-C30:0 primary alcohols. GFP-fusion protein injection assay showed that the three encoded TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The transcriptional expression of the three TaFAR genes was induced by cold, salt, drought and ABA. Low air humidity led to increased expression of TaFAR genes and elevated wax accumulation in wheat leaves. Collectively, these data suggest that TaFAR2, TaFAR3 and TaFAR4 encode active alcohol-forming FARs involved in the synthesis of primary alcohol in wheat leaf and the response to environmental stresses. PMID:27112792

  1. Disorders of lipid metabolism in nephrotic syndrome: mechanisms and consequences.

    PubMed

    Vaziri, Nosratola D

    2016-07-01

    Nephrotic syndrome results in hyperlipidemia and profound alterations in lipid and lipoprotein metabolism. Serum cholesterol, triglycerides, apolipoprotein B (apoB)-containing lipoproteins (very low-density lipoprotein [VLDL], immediate-density lipoprotein [IDL], and low-density lipoprotein [LDL]), lipoprotein(a) (Lp[a]), and the total cholesterol/high-density lipoprotein (HDL) cholesterol ratio are increased in nephrotic syndrome. This is accompanied by significant changes in the composition of various lipoproteins including their cholesterol-to-triglyceride, free cholesterol-to-cholesterol ester, and phospholipid-to-protein ratios. These abnormalities are mediated by changes in the expression and activities of the key proteins involved in the biosynthesis, transport, remodeling, and catabolism of lipids and lipoproteins including apoproteins A, B, C, and E; 3-hydroxy-3-methylglutaryl-coenzyme A reductase; fatty acid synthase; LDL receptor; lecithin cholesteryl ester acyltransferase; acyl coenzyme A cholesterol acyltransferase; HDL docking receptor (scavenger receptor class B, type 1 [SR-B1]); HDL endocytic receptor; lipoprotein lipase; and hepatic lipase, among others. The disorders of lipid and lipoprotein metabolism in nephrotic syndrome contribute to the development and progression of cardiovascular and kidney disease. In addition, by limiting delivery of lipid fuel to the muscles for generation of energy and to the adipose tissues for storage of energy, changes in lipid metabolism contribute to the reduction of body mass and impaired exercise capacity. This article provides an overview of the mechanisms, consequences, and treatment of lipid disorders in nephrotic syndrome. PMID:27165836

  2. Endothelial lipase modulates pressure overload-induced heart failure through alternative pathway for fatty acid uptake.

    PubMed

    Nakajima, Hideto; Ishida, Tatsuro; Satomi-Kobayashi, Seimi; Mori, Kenta; Hara, Tetsuya; Sasaki, Naoto; Yasuda, Tomoyuki; Toh, Ryuji; Tanaka, Hidekazu; Kawai, Hiroya; Hirata, Ken-ichi

    2013-05-01

    Lipoprotein lipase has been considered as the only enzyme capable of generating lipid-derived fatty acids for cardiac energy. Endothelial lipase is another member of the triglyceride lipase family and hydrolyzes high-density lipoproteins. Although endothelial lipase is expressed in the heart, its function remains unclear. We assessed the role of endothelial lipase in the genesis of heart failure. Pressure overload-induced cardiac hypertrophy was generated in endothelial lipase(-/-) and wild-type mice by ascending aortic banding. Endothelial lipase expression in cardiac tissues was markedly elevated in the early phase of cardiac hypertrophy in wild-type mice, whereas lipoprotein lipase expression was significantly reduced. Endothelial lipase(-/-) mice showed more severe systolic dysfunction with left-ventricular dilatation compared with wild-type mice in response to pressure overload. The expression of mitochondrial fatty acid oxidation-related genes, such as carnitine palmitoyltransferase-1 and medium-chain acyl coenzyme A dehydrogenase, was significantly lower in the heart of endothelial lipase(-/-) mice than in wild-type mice. Also, endothelial lipase(-/-) mice had lower myocardial adenosine triphosphate levels than wild-type mice after aortic banding. In cultured cardiomyocytes, endothelial lipase was upregulated by inflammatory stimuli, whereas lipoprotein lipase was downregulated. Endothelial lipase-overexpression in cardiomyocytes resulted in an upregulation of fatty acid oxidation-related enzymes and intracellular adenosine triphosphate accumulation in the presence of high-density lipoprotein. Endothelial lipase may act as an alternative candidate to provide fatty acids to the heart and regulate cardiac function. This effect seemed relevant particularly in the diseased heart, where lipoprotein lipase action is downregulated. PMID:23460280

  3. Ultra-fast simultaneous detection of obesity-related coenzymes in mice using microchip electrophoresis with a LIF detector.

    PubMed

    Lee, Hee Gu; Kumar, K S; Soh, Ju-Ryoun; Cha, Youn-Soo; Kang, Seong Ho

    2008-06-30

    Hepatic acyl-coenzyme A synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I) and acetyl coenzyme A carboxylase (ACC) are coenzymes associated with the genetic type of obesity in animal models. This paper reports the use of microchip electrophoresis (ME) with a laser-induced fluorescence (LIF) detector based on a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the amplified DNA fragments of these coenzymes (ACS, CPT-I and ACC) in the mRNA extracted from mice. DNA fragments ranging from 50 to 2652 bp were well resolved using this procedure with a running buffer (1x TBE), 0.5% polyvinylpyrrolidone (M(r) 1,000,000) as the coating gel and 0.7% polyethyleneoxide (M(r) 8,000,000) as the sieving gel at pH 8.30. The separation of the three RT-PCR products was achieved by ME in a single-run within 17 s using programmed field strength gradients (PFSG) (470 V cm(-1) for 9 s, 205.8 V cm(-1) for 2 s, 411.6 V cm(-1) for 4 s, 117.6 V cm(-1) for 2 s and 470.4V cm(-1) for 8 s). The ME-PFSG method was found to be 4 times faster than the method using a constant field and 138 times faster than slab gel electrophoresis. Moreover, the amplified RT-PCR products of the obesity-related coenzymes in C57BL/6J mice were analyzed using only sub-micro liter samples. PMID:18539180

  4. Proteomic analysis of formalin-fixed paraffin-embedded glomeruli suggests depletion of glomerular filtration barrier proteins in two-kidney, one-clip hypertensive rats

    PubMed Central

    Finne, Kenneth; Vethe, Heidrun; Skogstrand, Trude; Leh, Sabine; Dahl, Tone D.; Tenstad, Olav; Berven, Frode S.; Reed, Rolf K.; Vikse, Bjørn Egil

    2014-01-01

    Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. PMID:25129444

  5. Reducing Isozyme Competition Increases Target Fatty Acid Accumulation in Seed Triacylglycerols of Transgenic Arabidopsis1[OPEN

    PubMed Central

    van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D.; Browse, John

    2015-01-01

    One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies. PMID:25739701

  6. Inhibition by propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia.

    PubMed

    Coude, F X; Sweetman, L; Nyhan, W L

    1979-12-01

    In the search for the mechanism by which hyperammonemia complicates propionic and methylmalonic acidemia the effects of a series of acyl-coenzyme A (CoA) derivatives were studied on the activity of N-acetylglutamate synthetase in rat liver mitochondria using acetyl-CoA as substrate. Propionyl-CoA was found to be a competitive inhibitor. The inhibition constant of 0.71 mM is in the range of concentrations of propionate found in the serum of patients with propionic and methylmalonic acidemia. Propionyl-CoA was also found to be a substrate for N-acetylglutamate synthetase, forming N-propionylglutamate. This compound was a weak activator of rat liver carbamoylphosphate synthetase; the activation constant was 1.1 mM as compared with 0.12 mM for N-acetylglutamate. A decreased level of N-acetylglutamate in liver mitochondria that would follow inhibition of N-acetylglutamate synthetase by propionyl-CoA would be expected to lead to hyperammonemia. Methylmalonyl-CoA, tiglyl-CoA, and isovaleryl-CoA at a concentration of 3 mM caused 30-70% inhibition of N-acetylglutamate synthetase. 3the latter two compounds are readily detoxified by the formation of N-acylglycine conjugates in liver, which may prevent large accumulations and could explain why hyperammonemia is not characteristic of patients with beta-ketothiolase deficiency or isovaleric acidemia in whom these compounds would be expected to be elevated. PMID:500823

  7. Metabolic Regulation as a Consequence of Anaerobic 5-Methylthioadenosine Recycling in Rhodospirillum rubrum

    PubMed Central

    North, Justin A.; Sriram, Jaya; Chourey, Karuna; Ecker, Christopher D.; Sharma, Ritin; Wildenthal, John A.; Hettich, Robert L.

    2016-01-01

    ABSTRACT Rhodospirillum rubrum possesses a novel oxygen-independent, aerobic methionine salvage pathway (MSP) for recycling methionine from 5-methylthioadenosine (MTA), the MTA-isoprenoid shunt. This organism can also metabolize MTA as a sulfur source under anaerobic conditions, suggesting that the MTA-isoprenoid shunt may also function anaerobically as well. In this study, deep proteomics profiling, directed metabolite analysis, and reverse transcriptase quantitative PCR (RT-qPCR) revealed metabolic changes in response to anaerobic growth on MTA versus sulfate as sole sulfur source. The abundance of protein levels associated with methionine transport, cell motility, and chemotaxis increased in the presence of MTA over that in the presence of sulfate. Purine salvage from MTA resulted primarily in hypoxanthine accumulation and a decrease in protein levels involved in GMP-to-AMP conversion to balance purine pools. Acyl coenzyme A (acyl-CoA) metabolic protein levels for lipid metabolism were lower in abundance, whereas poly-β-hydroxybutyrate synthesis and storage were increased nearly 10-fold. The known R. rubrum aerobic MSP was also shown to be upregulated, to function anaerobically, and to recycle MTA. This suggested that other organisms with gene homologues for the MTA-isoprenoid shunt may also possess a functioning anaerobic MSP. In support of our previous findings that ribulose-1,5-carboxylase/oxygenase (RubisCO) is required for an apparently purely anaerobic MSP, RubisCO transcript and protein levels both increased in abundance by over 10-fold in cells grown anaerobically on MTA over those in cells grown on sulfate, resulting in increased intracellular RubisCO activity. These results reveal for the first time global metabolic responses as a consequence of anaerobic MTA metabolism compared to using sulfate as the sulfur source. PMID:27406564

  8. Salusin-β induces foam cell formation and monocyte adhesion in human vascular smooth muscle cells via miR155/NOX2/NFκB pathway

    PubMed Central

    Sun, Hai-Jian; Zhao, Ming-Xia; Liu, Tong-Yan; Ren, Xing-Sheng; Chen, Qi; Li, Yue-Hua; Kang, Yu-Ming; Zhu, Guo-Qing

    2016-01-01

    Vascular smooth muscle cells (VSMCs) are indispensible components in foam cell formation. Salusin-β is a stimulator in the progression of atherosclerosis. Here, we showed that salusin-β increased foam cell formation evidenced by accumulation of lipid droplets and intracellular cholesterol content, and promoted monocyte adhesion in human VSMCs. Salusin-β increased the expressions and activity of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1) and vascular cell adhesion molecule-1 (VCAM-1) in VSMCs. Silencing of ACAT-1 abolished the salusin-β-induced lipid accumulation, and silencing of VCAM-1 prevented the salusin-β-induced monocyte adhesion in VSMCs. Salusin-β caused p65-NFκB nuclear translocation and increased p65 occupancy at the ACAT-1 and VCAM-1 promoter. Inhibition of NFκB with Bay 11-7082 prevented the salusin-β-induced ACAT-1 and VCAM-1 upregulation, foam cell formation and monocyte adhesion in VSMCs. Scavenging ROS, inhibiting NADPH oxidase or knockdown of NOX2 abolished the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation and monocyte adhesion in VSMCs. Salusin-β increased miR155 expression, and knockdown of miR155 prevented the effects of salusin-β on ACAT-1 and VCAM-1 expressions, p65-NFκB nuclear translocation, lipid accumulation, monocyte adhesion and ROS production in VSMCs. These results indicate that salusin-β induces foam formation and monocyte adhesion via miR155/NOX2/NFκB-mediated ACAT-1 and VCAM-1 expressions in VSMCs. PMID:27004848

  9. Decreased liver triglyceride content in adult rats exposed to protein restriction during gestation and lactation: role of hepatic triglyceride utilization.

    PubMed

    Qasem, Rani J; Li, Jing; Tang, Hee Man; Browne, Veron; Mendez-Garcia, Claudia; Yablonski, Elizabeth; Pontiggia, Laura; D'Mello, Anil P

    2015-04-01

    We have previously demonstrated that protein restriction throughout gestation and lactation reduces liver triglyceride content in adult rat offspring. However, the mechanisms mediating the decrease in liver triglyceride content are not understood. The aim of the current study was to use a new group of pregnant animals and their offspring and determine the contribution of increased triglyceride utilization via the hepatic fatty-acid oxidation and triglyceride secretory pathways to the reduction in liver triglyceride content. Pregnant Sprague-Dawley rats received either a control or a low protein diet throughout pregnancy and lactation. Pups were weaned onto laboratory chow on day 28 and killed on day 65. Liver triglyceride content was reduced in male, but not female, low-protein offspring, both in the fed and fasted states. The reduction was accompanied by a trend towards higher liver carnitine palmitoyltransferase-1a activity, suggesting increased fatty-acid transport into the mitochondrial matrix. However, medium-chain acyl coenzyme A dehydrogenase activity within the mitochondrial matrix, expression of nuclear peroxisome proliferator activated receptor-α, and plasma levels of β-hydroxybutyrate were similar between low protein and control offspring, indicating a lack of change in fatty-acid oxidation. Hepatic triglyceride secretion, assessed by blocking peripheral triglyceride utilization and measuring serum triglyceride accumulation rate, and the activity of microsomal transfer protein, were similar between low protein and control offspring. Because enhanced triglyceride utilization is not a significant contributor, the decrease in liver triglyceride content in male low-protein offspring is likely due to alterations in liver fatty-acid transport or triglyceride biosynthesis. PMID:25641378

  10. Systematic Mutational Analysis of Histidine Kinase Genes in the Nosocomial Pathogen Stenotrophomonas maltophilia Identifies BfmAK System Control of Biofilm Development.

    PubMed

    Zheng, Liu; Wang, Fang-Fang; Ren, Bao-Zhen; Liu, Wei; Liu, Zhong; Qian, Wei

    2016-04-01

    The Gram-negative bacterium Stenotrophomonas maltophilialives in diverse ecological niches. As a result of its formidable capabilities of forming biofilm and its resistance to multiple antibiotic agents, the bacterium is also a nosocomial pathogen of serious threat to the health of patients whose immune systems are suppressed or compromised. Besides the histidine kinase RpfC, the two-component signal transduction system (TCS), which is the canonical regulatory machinery used by most bacterial pathogens, has never been experimentally investigated inS. maltophilia Here, we annotated 62 putative histidine kinase genes in the S. maltophilia genome and successfully obtained 51 mutants by systematical insertional inactivation. Phenotypic characterization identified a series of mutants with deficiencies in bacterial growth, swimming motility, and biofilm development. A TCS, named here BfmA-BfmK (Smlt4209-Smlt4208), was genetically confirmed to regulate biofilm formation inS. maltophilia Together with interacting partner prediction and chromatin immunoprecipitation screens, six candidate promoter regions bound by BfmA in vivo were identified. We demonstrated that, among them, BfmA acts as a transcription factor that binds directly to the promoter regions of bfmA-bfmK and Smlt0800(acoT), a gene encoding an acyl coenzyme A thioesterase that is associated with biofilm development, and positively controls their transcription. Genome-scale mutational analyses of histidine kinase genes and functional dissection of BfmK-BfmA regulation in biofilm provide genetic information to support more in-depth studies on cellular signaling inS. maltophilia, in the context of developing novel approaches to fight this important bacterial pathogen. PMID:26873318

  11. Aflavinines and other antiinsectan metabolites from the ascostromata of Eupenicillium crustaceum and related species.

    PubMed

    Wang, H J; Gloer, J B; Wicklow, D T; Dowd, P F

    1995-12-01

    This report describes the distribution of antiinsectan metabolites present in sclerotioid ascostromata produced by representative strains of Eupenicillium crustaceum and fungal taxa that are considered to be closely related. The hexane and chloroform extracts of E. crustaceum NRRL 3332 displayed significant antiinsectan activity in assays against the corn earworm, Helicoverpa zea. The major metabolite accounting for this antiinsectan activity was a known aflavinine analog, 10,23-dihydro-24,25-dehydroaflavinine, occurring at approximately 2.8 mg/g of dry ascostromata. In dietary assays at ca. 3,000 ppm, a 79% reduction in weight gain and a 42% reduction in feeding rate were observed in H. zea and Carpophilus hemipterus larvae, respectively. A new aflavinine analog, 10,23,24,25-tetrahydro-24-hydroxyaflavinine, was also identified. These aflavinine compounds are the first to be reported from a fungal genus other than Aspergillus. New macrophorin-type metabolites accounted for the antiinsectan activity of ascostromata produced by E. crustaceum NRRL 22307, which produced no aflavinines, while Eupenicillium molle NRRL 13062 produced both aflavinines and macrophorins. Sclerotia produced by Penicillium gladioli NRRL 938, NRRL 939, and QM 2743, a fungus reported to be conspecific with the anamorph of E. crustaceum, produced neither aflavinines nor macrophorins. Eupenicillium reticulisporum NRRL 3446 produced the aflavinine analog 10,23-dihydro-24,25-dehydroaflavinine and an unrelated compound called pyripyropene A, a potent inhibitor of acyl-coenzyme A-cholesterol acyltransferase. Eupenicillium abidjanum NRRL 5809, reported to be conspecific with E. reticulisporum, produced neither of these compounds. The Eupenicillium species that produced aflavinines are also known for their ability to grow rapidly with reduced water activity. PMID:8534106

  12. Estrogen Decreases Atherosclerosis In Part By Reducing Hepatic Acyl-CoA:Cholesterol Acyltransferase 2 (ACAT2) In Monkeys

    PubMed Central

    Kavanagh, Kylie; Davis, Matthew A.; Zhang, Li; Wilson, Martha D.; Register, Thomas C.; Adams, Michael R.; Rudel, Lawrence L.; Wagner, Janice D.

    2009-01-01

    Objective Estrogens decrease atherosclerosis progression, mediated in part through changes in plasma lipids and lipoproteins. This study aimed to determine estrogen-induced changes in hepatic cholesterol metabolism, plasma lipoproteins, and the relationship of these changes to atherosclerosis extent. Methods and Results Ovariectomized monkeys (n=34) consumed atherogenic diets for 30 months which contained either no hormones (control, n=17) or conjugated equine estrogens (CEE, n=17) at a human dose equivalent of 0.625 mg/d. Hepatic cholesterol content, low-density lipoprotein (LDL) receptor expression, cholesterol 7α-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and expression levels were determined. CEE treatment resulted in lower plasma concentrations of very-low- and intermediate density lipoprotein cholesterol (V+IDLC; p=0.01), smaller LDL particles (p=0.002) and 50% lower hepatic cholesterol content (total, free and esterified; p<0.05 for all). Total ACAT activity was significantly lower (p=0.01), explained primarily by reductions in the activity of ACAT2. Estrogen regulation of enzymatic activity was at the protein level as both ACAT1 and 2 protein, but not mRNA levels, were lower (p=0.02 and <0.0001, respectively). ACAT2 activity was significantly associated with hepatic total cholesterol, plasma V+IDLC cholesterol, and atherosclerosis. Conclusions Atheroprotective effects of estrogen therapy may be related to reduced hepatic secretion of ACAT2-derived cholesteryl esters in plasma lipoproteins. Condensed Abstract Estrogen inhibits atherogenesis. We demonstrate in ovariectorized monkeys that estrogen therapy led to lower hepatic and circulating lipoprotein cholesterol, and lower ACAT2 protein and associated activity levels as compared to controls. Hepatic ACAT2 activity was highly correlated with, and was an independent predictor of coronary artery atherosclerosis extent. PMID:19759374

  13. Aflavinines and other antiinsectan metabolites from the ascostromata of Eupenicillium crustaceum and related species.

    PubMed Central

    Wang, H J; Gloer, J B; Wicklow, D T; Dowd, P F

    1995-01-01

    This report describes the distribution of antiinsectan metabolites present in sclerotioid ascostromata produced by representative strains of Eupenicillium crustaceum and fungal taxa that are considered to be closely related. The hexane and chloroform extracts of E. crustaceum NRRL 3332 displayed significant antiinsectan activity in assays against the corn earworm, Helicoverpa zea. The major metabolite accounting for this antiinsectan activity was a known aflavinine analog, 10,23-dihydro-24,25-dehydroaflavinine, occurring at approximately 2.8 mg/g of dry ascostromata. In dietary assays at ca. 3,000 ppm, a 79% reduction in weight gain and a 42% reduction in feeding rate were observed in H. zea and Carpophilus hemipterus larvae, respectively. A new aflavinine analog, 10,23,24,25-tetrahydro-24-hydroxyaflavinine, was also identified. These aflavinine compounds are the first to be reported from a fungal genus other than Aspergillus. New macrophorin-type metabolites accounted for the antiinsectan activity of ascostromata produced by E. crustaceum NRRL 22307, which produced no aflavinines, while Eupenicillium molle NRRL 13062 produced both aflavinines and macrophorins. Sclerotia produced by Penicillium gladioli NRRL 938, NRRL 939, and QM 2743, a fungus reported to be conspecific with the anamorph of E. crustaceum, produced neither aflavinines nor macrophorins. Eupenicillium reticulisporum NRRL 3446 produced the aflavinine analog 10,23-dihydro-24,25-dehydroaflavinine and an unrelated compound called pyripyropene A, a potent inhibitor of acyl-coenzyme A-cholesterol acyltransferase. Eupenicillium abidjanum NRRL 5809, reported to be conspecific with E. reticulisporum, produced neither of these compounds. The Eupenicillium species that produced aflavinines are also known for their ability to grow rapidly with reduced water activity. PMID:8534106

  14. Ethylene Glycol Monomethyl Ether–Induced Toxicity Is Mediated through the Inhibition of Flavoprotein Dehydrogenase Enzyme Family

    PubMed Central

    Takei, Makoto; Ando, Yosuke; Saitoh, Wataru; Tanimoto, Tomoe; Kiyosawa, Naoki; Manabe, Sunao; Sanbuissho, Atsushi; Okazaki, Osamu; Iwabuchi, Haruo; Yamoto, Takashi; Adam, Klaus-Peter; Weiel, James E.; Ryals, John A.; Milburn, Michael V.; Guo, Lining

    2010-01-01

    Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage. PMID:20616209

  15. Targeted enhancement of glutamate-to-γ-aminobutyrate conversion in Arabidopsis seeds affects carbon-nitrogen balance and storage reserves in a development-dependent manner.

    PubMed

    Fait, Aaron; Nesi, Adriano Nunes; Angelovici, Ruthie; Lehmann, Martin; Pham, Phuong Anh; Song, Luhua; Haslam, Richard P; Napier, Johnathan A; Galili, Gad; Fernie, Alisdair R

    2011-11-01

    In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca(2+)-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation. PMID:21921115

  16. Operon for Biosynthesis of Lipstatin, the Beta-Lactone Inhibitor of Human Pancreatic Lipase

    PubMed Central

    Bai, Tingli; Zhang, Daozhong; Lin, Shuangjun; Long, Qingshan; Wang, Yemin; Ou, Hongyu; Kang, Qianjin; Deng, Zixin; Liu, Wen

    2014-01-01

    Lipstatin, isolated from Streptomyces toxytricini as a potent and selective inhibitor of human pancreatic lipase, is a precursor for tetrahydrolipstatin (also known as orlistat, Xenical, and Alli), the only FDA-approved antiobesity medication for long-term use. Lipstatin features a 2-hexyl-3,5-dihydroxy-7,10-hexadecadienoic-β-lactone structure with an N-formyl-l-leucine group attached as an ester to the 5-hydroxy group. It has been suggested that the α-branched 3,5-dihydroxy fatty acid β-lactone moiety of lipstatin in S. toxytricini is derived from Claisen condensation between two fatty acid substrates, which are derived from incomplete oxidative degradation of linoleic acid based on feeding experiments. In this study, we identified a six-gene operon (lst) that was essential for the biosynthesis of lipstatin by large-deletion, complementation, and single-gene knockout experiments. lstA, lstB, and lstC, which encode two β-ketoacyl–acyl carrier protein synthase III homologues and an acyl coenzyme A (acyl-CoA) synthetase homologue, were indicated to be responsible for the generation of the α-branched 3,5-dihydroxy fatty acid backbone. Subsequently, the nonribosomal peptide synthetase (NRPS) gene lstE and the putative formyltransferase gene lstF were involved in decoration of the α-branched 3,5-dihydroxy fatty acid chain with an N-formylated leucine residue. Finally, the 3β-hydroxysteroid dehydrogenase-homologous gene lstD might be responsible for the reduction of the β-keto group of the biosynthetic intermediate, thereby facilitating the formation of the unique β-lactone ring. PMID:25239907

  17. Functional Promiscuity of Two Divergent Paralogs of Type III Plant Polyketide Synthases.

    PubMed

    Pandith, Shahzad A; Dhar, Niha; Rana, Satiander; Bhat, Wajid Waheed; Kushwaha, Manoj; Gupta, Ajai P; Shah, Manzoor A; Vishwakarma, Ram; Lattoo, Surrinder K

    2016-08-01

    Plants effectively defend themselves against biotic and abiotic stresses by synthesizing diverse secondary metabolites, including health-protective flavonoids. These display incredible chemical diversity and ubiquitous occurrence and confer impeccable biological and agricultural applications. Chalcone synthase (CHS), a type III plant polyketide synthase, is critical for flavonoid biosynthesis. It catalyzes acyl-coenzyme A thioesters to synthesize naringenin chalcone through a polyketidic intermediate. The functional divergence among the evolutionarily generated members of a gene family is pivotal in driving the chemical diversity. Against this backdrop, this study was aimed to functionally characterize members of the CHS gene family from Rheum emodi, an endangered and endemic high-altitude medicinal herb of northwestern Himalayas. Two full-length cDNAs (1,179 bp each), ReCHS1 and ReCHS2, encoding unique paralogs were isolated and characterized. Heterologous expression and purification in Escherichia coli, bottom-up proteomic characterization, high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, and enzyme kinetic studies using five different substrates confirmed their catalytic potential. Phylogenetic analysis revealed the existence of higher synonymous mutations in the intronless divergents of ReCHS. ReCHS2 displayed significant enzymatic efficiency (Vmax/Km) with different substrates. There were significant spatial and altitudinal variations in messenger RNA transcript levels of ReCHSs correlating positively with metabolite accumulation. Furthermore, the elicitations in the form of methyl jasmonate, salicylic acid, ultraviolet B light, and wounding, chosen on the basis of identified cis-regulatory promoter elements, presented considerable differences in the transcript profiles of ReCHSs. Taken together, our results demonstrate differential propensities of CHS paralogs in terms of the accumulation of flavonoids and

  18. Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

    PubMed

    Lassila, Toni; Hokkanen, Juho; Aatsinki, Sanna-Mari; Mattila, Sampo; Turpeinen, Miia; Tolonen, Ari

    2015-12-21

    Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-β-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI). PMID:26558897

  19. Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production

    PubMed Central

    2012-01-01

    Background Wax ester synthases (WSs) can synthesize wax esters from alcohols and fatty acyl coenzyme A thioesters. The knowledge of the preferred substrates for each WS allows the use of yeast cells for the production of wax esters that are high-value materials and can be used in a variety of industrial applications. The products of WSs include fatty acid ethyl esters, which can be directly used as biodiesel. Results Here, heterologous WSs derived from five different organisms were successfully expressed and evaluated for their substrate preference in Saccharomyces cerevisiae. We investigated the potential of the different WSs for biodiesel (that is, fatty acid ethyl esters) production in S. cerevisiae. All investigated WSs, from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter arcticus 273-4, have different substrate specificities, but they can all lead to the formation of biodiesel. The best biodiesel producing strain was found to be the one expressing WS from M. hydrocarbonoclasticus DSM 8798 that resulted in a biodiesel titer of 6.3 mg/L. To further enhance biodiesel production, acetyl coenzyme A carboxylase was up-regulated, which resulted in a 30% increase in biodiesel production. Conclusions Five WSs from different species were functionally expressed and their substrate preference characterized in S. cerevisiae, thus constructing cell factories for the production of specific kinds of wax ester. WS from M. hydrocarbonoclasticus showed the highest preference for ethanol compared to the other WSs, and could permit the engineered S. cerevisiae to produce biodiesel. PMID:22364438

  20. Human urotensin II promotes hypertension and atherosclerotic cardiovascular diseases.

    PubMed

    Watanabe, Takuya; Arita, Shigeko; Shiraishi, Yuji; Suguro, Toshiaki; Sakai, Tetsuo; Hongo, Shigeki; Miyazaki, Akira

    2009-01-01

    Human urotensin II (U-II), the most potent vasoconstrictor undecapeptide identified to date, and its receptor (UT) are involved in the pathogenesis of systemic and pulmonary hypertension. Here, we review recent advances in our understanding of the pathophysiology of U-II with particular reference to its role in atherosclerotic cardiovascular diseases. Single-nucleotide polymorphisms of U-II gene (S89N) are associated with onset of essential hypertension, type II diabetes mellitus, and insulin resistance in the Asian population. Plasma U-II levels are elevated in patients with vascular endothelial dysfunction-related diseases such as essential hypertension, diabetes mellitus, atherosclerosis, ischemic heart disease, and heart failure. Chronic infusion of U-II enhances atherosclerotic lesions in the aorta in apolipoprotein E-knockout mice. In human atherosclerotic plaques from the aorta and coronary and carotid arteries, U-II is expressed at high levels in endothelial cells (ECs) and lymphocytes, whereas UT is expressed at high levels in vascular smooth muscle cells (VSMCs), ECs, monocytes, and macrophages. U-II stimulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in human ECs as chemoattractant for monocytes, and accelerates foam cell formation by up-regulation of acyl-coenzyme A:cholesterol acyltransferase-1 in human monocyte-derived macrophages. U-II produces reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate oxidase activation in human VSMCs, and stimulates VSMC proliferation with synergistic effects when combined with ROS, oxidized LDL, and serotonin. Clinical studies demonstrated increased plasma U-II levels in accordance with the severity of carotid atherosclerosis in patients with essential hypertension and that of coronary artery lesions in patients with ischemic heart disease. Here, we summarize the key roles of U-II in progression of hypertension and atherosclerotic cardiovascular diseases

  1. Skeletal Muscle Magnetic Resonance Imaging of the Lower Limbs in Late-onset Lipid Storage Myopathy with Electron Transfer Flavoprotein Dehydrogenase Gene Mutations

    PubMed Central

    Liu, Xin-Yi; Jin, Ming; Wang, Zhi-Qiang; Wang, Dan-Ni; He, Jun-Jie; Lin, Min-Ting; Fu, Hong-Xia; Wang, Ning

    2016-01-01

    Background: Lipid storage myopathy (LSM) is a genetically heterogeneous group with variable clinical phenotypes. Late-onset multiple acyl-coenzyme A dehydrogenation deficiency (MADD) is a rather common form of LSM in China. Diagnosis and clinical management of it remain challenging, especially without robust muscle biopsy result and genetic detection. As the noninvasion and convenience, muscle magnetic resonance imaging (MRI) is a helpful assistant, diagnostic tool for neuromuscular disorders. However, the disease-specific MRI patterns of muscle involved and its diagnostic value in late-onset MADD have not been systematic analyzed. Methods: We assessed the MRI pattern and fat infiltration degree of the lower limb muscles in 28 late-onset MADD patients, combined with detailed clinical features and gene spectrum. Fat infiltration degree of the thigh muscle was scored while that of gluteus was described as obvious or not. Associated muscular atrophy was defined as obvious muscle bulk reduction. Results: The mean scores were significantly different among the anterior, medial, and posterior thigh muscle groups. The mean of fat infiltration scores on posterior thigh muscle group was significantly higher than either anterior or medial thigh muscle group (P < 0.001). Moreover, the mean score on medial thigh muscle group was significantly higher than that of anterior thigh muscle group (P < 0.01). About half of the patients displayed fat infiltration and atrophy in gluteus muscles. Of 28 patients, 12 exhibited atrophy in medial and/or posterior thigh muscle groups, especially in posterior thigh muscle group. Muscle edema pattern was not found in all the patients. Conclusions: Late-onset MADD patients show a typical muscular imaging pattern of fat infiltration and atrophy on anterior, posterior, and medial thigh muscle groups, with major involvement of posterior thigh muscle group and gluteus muscles and a sparing involvement of anterior thigh compartment. Our findings also

  2. Computational analysis of a novel mutation in ETFDH gene highlights its long-range effects on the FAD-binding motif

    PubMed Central

    2011-01-01

    Background Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an autosomal recessive disease caused by the defects in the mitochondrial electron transfer system and the metabolism of fatty acids. Recently, mutations in electron transfer flavoprotein dehydrogenase (ETFDH) gene, encoding electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO) have been reported to be the major causes of riboflavin-responsive MADD. To date, no studies have been performed to explore the functional impact of these mutations or their mechanism of disrupting enzyme activity. Results High resolution melting (HRM) analysis and sequencing of the entire ETFDH gene revealed a novel mutation (p.Phe128Ser) and the hotspot mutation (p.Ala84Thr) from a patient with MADD. According to the predicted 3D structure of ETF:QO, the two mutations are located within the flavin adenine dinucleotide (FAD) binding domain; however, the two residues do not have direct interactions with the FAD ligand. Using molecular dynamics (MD) simulations and normal mode analysis (NMA), we found that the p.Ala84Thr and p.Phe128Ser mutations are most likely to alter the protein structure near the FAD binding site as well as disrupt the stability of the FAD binding required for the activation of ETF:QO. Intriguingly, NMA revealed that several reported disease-causing mutations in the ETF:QO protein show highly correlated motions with the FAD-binding site. Conclusions Based on the present findings, we conclude that the changes made to the amino acids in ETF:QO are likely to influence the FAD-binding stability. PMID:22013910

  3. First report about the mode of action of combined butafosfan and cyanocobalamin on hepatic metabolism in nonketotic early lactating cows.

    PubMed

    Kreipe, L; Deniz, A; Bruckmaier, R M; van Dorland, H A

    2011-10-01

    The primary aim was to investigate the effect of combined butafosfan and cyanocobalamin on liver metabolism in early lactating cows through mRNA expression measurements of genes encoding 31 enzymes and transport proteins of major metabolic processes in the liver using 16 multiparous early lactating dairy cows. The treatments included i.v. injection of 10 mL/100 kg of body weight combined butafosfan and cyanocobalamin (TG, n = 8) on 3 d consecutively at 25 ± 3 d in milk or injection with physiological saline solution similarly applied (CG, n = 8). Results include a higher daily milk production for TG cows (41.1 ± 0.9 kg, mean ± SEM) compared with CG cows (39.5 ± 0.7 kg). In plasma, the concentration of inorganic phosphorus was lower in the TG cows (1.25 ± 0.08 mmol/L) after the treatment than in the CG cows (1.33 ± 0.07 mmol/L). The plasma β-hydroxybutyrate concentration was 0.65 ± 0.13 mmol/L for all cows before the treatment, and remained unaffected post treatment. The unique result was that in the liver, the mRNA abundance of acyl-coenzyme A synthetase long-chain family member 1, involved in fatty acid oxidation and biosynthesis, was lower across time points after the treatment for TG compared with CG cows (17.5 ± 0.15 versus 18.1 ± 0.24 cycle threshold, log(2), respectively). In conclusion, certain effects of combined butafosfan and cyanocobalamin were observed on mRNA abundance of a gene in the liver of nonketotic early lactating cows. PMID:21943742

  4. Therapeutic potential of chalcones as cardiovascular agents.

    PubMed

    Mahapatra, Debarshi Kar; Bharti, Sanjay Kumar

    2016-03-01

    Cardiovascular diseases are the leading cause of death affecting 17.3 million people across the globe and are estimated to affect 23.3 million people by year 2030. In recent years, about 7.3 million people died due to coronary heart disease, 9.4 million deaths due to high blood pressure and 6.2 million due to stroke, where obesity and atherosclerotic progression remain the chief pathological factors. The search for newer and better cardiovascular agents is the foremost need to manage cardiac patient population across the world. Several natural and (semi) synthetic chalcones deserve the credit of being potential candidates to inhibit various cardiovascular, hematological and anti-obesity targets like angiotensin converting enzyme (ACE), cholesteryl ester transfer protein (CETP), diacylglycerol acyltransferase (DGAT), acyl-coenzyme A: cholesterol acyltransferase (ACAT), pancreatic lipase (PL), lipoprotein lipase (LPL), calcium (Ca(2+))/potassium (K(+)) channel, COX-1, TXA2 and TXB2. In this review, a comprehensive study of chalcones, their therapeutic targets, structure activity relationships (SARs), mechanisms of actions (MOAs) have been discussed. Chemically diverse chalcone scaffolds, their derivatives including structural manipulation of both aryl rings, replacement with heteroaryl scaffold(s) and hybridization through conjugation with other pharmacologically active scaffold have been highlighted. Chalcones which showed promising activity and have a well-defined MOAs, SARs must be considered as prototype for the design and development of potential anti-hypertensive, anti-anginal, anti-arrhythmic and cardioprotective agents. With the knowledge of these molecular targets, structural insights and SARs, this review may be helpful for (medicinal) chemists to design more potent, safe, selective and cost effective chalcone derivatives as potential cardiovascular agents. PMID:26876916

  5. Newer antiatherosclerosis treatment strategies

    PubMed Central

    Aggarwal, Amitesh; Singh, Safal

    2011-01-01

    Atherosclerosis has been a target of much clinical and molecular research. As a result of this extensive research, it is amply clear that atherogenesis is a multifactorial process involving an interplay of metabolic, immune and inflammatory mechanisms. Antiatherosclerotic strategies are today aiming for a multipronged approach targeting each arm of this multifactorial process. The newer agents under development can be divided into three broad categories: anti-inflammatory agents, modulators of intermediary metabolism and antiatherosclerosis vaccines. Potential targets for anti-inflammatory agents include inhibition of conversion of low-density lipoprotein (LDL) to oxidised LDL, blocking or downregulation of cell adhesion molecules, chemokine modulation and macrophage receptor blockade. Beyond inhibition of plaque formation, efforts are also ongoing to develop agents which stabilise the plaque by increasing its fibrous content and inhibiting its disruption. So far as research in the sphere of intermediary metabolism is concerned, the focus is now primarily on raising high-density lipoprotein and promoting reverse cholesterol transport; potential targets include cholesteryl ester transfer protein, liver X-receptor, lecithin cholesterol acyltransferase and high-density lipoprotein mimetics. Acyl-coenzymeA: cholesterol acyltransferase is another enzyme whose selective and differential inhibition is under active investigation. The concept of immunisation against a non-communicable disease such as atherosclerosis is still in its nascent stages. However, with increasing evidence to suggest the role of antigen-specific T-cell-mediated immunity in atherogenesis, this approach is potentially promising. Possible antigens under evaluation include oxidised LDL and its subparticles, heat-shock proteins and cholesteryl ester transfer protein. With cardiovascular disease being the single leading cause of death worldwide, the development of a safe and successful antiatherosclerosis

  6. Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Purves, Randy W; Ambrose, Stephen J; Clark, Shawn M; Stout, Jake M; Page, Jonathan E

    2015-02-01

    Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants. PMID:25553535

  7. Urinary metabolic profile predicts high-fat diet sensitivity in the C57Bl6/J mouse.

    PubMed

    Fedry, Juliette; Blais, Anne; Even, Patrick C; Piedcoq, Julien; Fromentin, Gilles; Gaudichon, Claire; Azzout-Marniche, Dalila; Tomé, Daniel

    2016-05-01

    To prevent the development of adiposity-associated metabolic diseases, early biomarkers are needed. Such markers could bring insight to understand the complexity of susceptibility to obesity. Urine and plasma metabolomics fingerprints have been successfully associated with metabolic dysfunctions. Fat resistance (FR) was found to be associated with higher urinary levels of acylglycines and leucine. However, no differences were observed before the diet switch. In this context, we aimed at characterizing metabolic signatures predictive of resistance or sensitivity to fat in the C57Bl6/J mouse model. Urinary metabolic profiles of FR (n=15) and fat sensitivity (FS) mice (n=14) were performed on liquid chromatography-mass spectrometry. Urinary and plasma metabolic profiles were first collected at baseline (during low-fat diet), then after 10weeks of high-fat (HF) feeding. Mice were sorted a posteriori into FS and FR based on their final adiposity. After HF feeding for 10weeks, FS mice tended to have lower plasma levels of β-hydroxybutyrate than FR ones. Urinary metabolic profiles showed that baseline levels of octanoylglycine, leucine and valine were significantly lower in FS mice. Moreover, expressions in the adipose tissue of Baat and Glyat mRNA were lower in FS than in FR mice. In muscle, mRNA encoding CaD and UbE2b tended to be lower in FS mice than in FR mice (P=.056 and P=.071, respectively). The data show that lower levels of urinary octanoylglycine, leucine and valine are potential predictive biomarkers of FS and could be related to a lower stimulation in adipose acyl-coenzyme A conjugation to glycine and to muscle protein breakdown. PMID:27133427

  8. The Mechanisms Underlying the Hypolipidaemic Effects of Grifola frondosa in the Liver of Rats.

    PubMed

    Ding, Yinrun; Xiao, Chun; Wu, Qingping; Xie, Yizhen; Li, Xiangmin; Hu, Huiping; Li, Liangqiu

    2016-01-01

    The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats, and of hyperlipidaemic rats treated with oral G. frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with G. frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acyl-coenzyme A: cholesterol acyltransferase (ACAT2), apolipoprotein B (ApoB), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC1) were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1) was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to identify 20 proteins differentially expressed in livers of rats treated with G. frondosa compared with untreated hyperlipidemic rats. Of these 20 proteins, seven proteins were down-regulated, and 13 proteins were up-regulated. These findings indicate that the hypolipidaemic effects of G. frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption, and catabolic pathways. G. frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, G. frondosa may produce both hypolipidaemic and anti-atherosclerotic effects, and potentially

  9. Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance.

    PubMed

    Luttgeharm, Kyle D; Chen, Ming; Mehra, Amit; Cahoon, Rebecca E; Markham, Jonathan E; Cahoon, Edgar B

    2015-10-01

    Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance

  10. Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system

    SciTech Connect

    Miyamoto, C.M.; Graham, A.D.; Boylan, M.; Evans, J.F.; Hasel, K.W.; Meighen, E.A.; Graham, A.F.

    1985-03-01

    DNA coding for the ..cap alpha.. and ..beta.. subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the ..cap alpha.. subunit as a hybridization probe, the authors identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the ..cap alpha.. and ..beta.. subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to the released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase ..cap alpha.. and ..beta.. subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.

  11. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

    PubMed

    Stout, Jake M; Boubakir, Zakia; Ambrose, Stephen J; Purves, Randy W; Page, Jonathan E

    2012-08-01

    The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids. PMID:22353623

  12. ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs.

    PubMed

    LaPensee, Christopher R; Mann, Jacqueline E; Rainey, William E; Crudo, Valentina; Hunt, Stephen W; Hammer, Gary D

    2016-05-01

    ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis. PMID:26986192

  13. Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines

    PubMed Central

    Olsen, Aaron; Chen, Yong; Ji, Qingzhou; Zhu, Guofeng; De Silva, Aruna Dharshan; Vilchèze, Catherine; Weisbrod, Torin; Li, Weimin; Xu, Jiayong; Larsen, Michelle; Zhang, Jinghang; Porcelli, Steven A.; Jacobs, William R.

    2016-01-01

    ABSTRACT Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4+ T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8+ T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis. The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4+ and CD8+ T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines. PMID:27247233

  14. High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells

    PubMed Central

    2011-01-01

    Background Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. Findings With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. Conclusions These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE. PMID:22163040

  15. Effects of dietary supplementation of coriander oil, in canola oil diets, on the metabolism of [1-(14)C] 18:3n-3 and [1-(14)C] 18:2n-6 in rainbow trout hepatocytes.

    PubMed

    Randall, K M; Drew, M D; Øverland, M; Østbye, T-K; Bjerke, M; Vogt, G; Ruyter, B

    2013-09-01

    The aim of this study was to investigate the effects of petroselinic acid, found in coriander oil, on the ability of rainbow trout hepatocytes to increase the production of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) from [1-(14)C] α-linolenic acid (18:3n-3; ALA) and to reduce the production of arachidonic acid (20:4n-6; ARA) from [1-(14)C] 18:2n-6. Addition of coriander oil increased the production of 22:6n-3, from [1-(14)C] 18:3n-3, at the 0.5 and 1.0% inclusion levels and reduced the conversion of [1-(14)C] 18:2n-6 to 20:4n-6. β-Oxidation was significantly increased at the 1.5% inclusion level for [1-(14)C] 18:2n-6, however β-oxidation for [1-(14)C] 18:3n-3 only showed an increasing trend. Acetate, a main breakdown product of fatty acids (FA) via peroxisomal β-oxidation, decreased three-fold for [1-(14)C] 18:2n-6 and nearly doubled for [1-(14)C] 18:3n-3 when coriander was added at a 1.5% inclusion level. Acyl coenzyme A oxidase (ACO) enzyme activity showed no significant differences between treatments. Relative gene expression of ∆6 desaturase decreased with addition of coriander oil compared to the control. The addition of petroselinic acid via coriander oil to vegetable oil (VO) based diets containing no fishmeal (FM) or fish oil (FO), significantly increased the production of anti-inflammatory precursor 22:6n-3 (P=0.011) and decreased pro-inflammatory precursor 20:4n-6 (P=0.023) in radiolabelled hepatocytes of rainbow trout. PMID:23867781

  16. Three TaFAR genes function in the biosynthesis of primary alcohols and the response to abiotic stresses in Triticum aestivum

    PubMed Central

    Wang, Meiling; Wang, Yong; Wu, Hongqi; Xu, Jing; Li, Tingting; Hegebarth, Daniela; Jetter, Reinhard; Chen, Letian; Wang, Zhonghua

    2016-01-01

    Cuticular waxes play crucial roles in protecting plants against biotic and abiotic stresses. They are complex mixtures of very-long-chain fatty acids and their derivatives, including C20–C32 fatty alcohols. Here, we report the identification of 32 FAR-like genes and the detailed characterization of TaFAR2, TaFAR3 and TaFAR4, wax biosynthetic genes encoding fatty acyl-coenzyme A reductase (FAR) in wheat leaf cuticle. Heterologous expression of the three TaFARs in wild-type yeast and mutated yeast showed that TaFAR2, TaFAR3 and TaFAR4 were predominantly responsible for the accumulation of C18:0, C28:0 and C24:0 primary alcohols, respectively. Transgenic expression of the three TaFARs in tomato fruit and Arabidopsis cer4 mutant led to increased production of C22:0–C30:0 primary alcohols. GFP-fusion protein injection assay showed that the three encoded TaFAR proteins were localized to the endoplasmic reticulum (ER), the site of wax biosynthesis. The transcriptional expression of the three TaFAR genes was induced by cold, salt, drought and ABA. Low air humidity led to increased expression of TaFAR genes and elevated wax accumulation in wheat leaves. Collectively, these data suggest that TaFAR2, TaFAR3 and TaFAR4 encode active alcohol-forming FARs involved in the synthesis of primary alcohol in wheat leaf and the response to environmental stresses. PMID:27112792

  17. Overexpression of a BAHD Acyltransferase, OsAt10, Alters Rice Cell Wall Hydroxycinnamic Acid Content and Saccharification1[C][W][OA

    PubMed Central

    Bartley, Laura E.; Peck, Matthew L.; Kim, Sung-Ryul; Ebert, Berit; Manisseri, Chithra; Chiniquy, Dawn M.; Sykes, Robert; Gao, Lingfang; Rautengarten, Carsten; Vega-Sánchez, Miguel E.; Benke, Peter I.; Canlas, Patrick E.; Cao, Peijian; Brewer, Susan; Lin, Fan; Smith, Whitney L.; Zhang, Xiaohan; Keasling, Jay D.; Jentoff, Rolf E.; Foster, Steven B.; Zhou, Jizhong; Ziebell, Angela; An, Gynheung; Scheller, Henrik V.; Ronald, Pamela C.

    2013-01-01

    Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a five-carbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed. PMID:23391577

  18. Fatty Acid Biosynthesis in Pseudomonas aeruginosa Is Initiated by the FabY Class of β-Ketoacyl Acyl Carrier Protein Synthases

    PubMed Central

    Yuan, Yanqiu; Sachdeva, Meena; Leeds, Jennifer A.

    2012-01-01

    The prototypical type II fatty acid synthesis (FAS) pathway in bacteria utilizes two distinct classes of β-ketoacyl synthase (KAS) domains to assemble long-chain fatty acids, the KASIII domain for initiation and the KASI/II domain for elongation. The central role of FAS in bacterial viability and virulence has stimulated significant effort toward developing KAS inhibitors, particularly against the KASIII domain of the β-acetoacetyl-acyl carrier protein (ACP) synthase FabH. Herein, we show that the opportunistic pathogen Pseudomonas aeruginosa does not utilize a FabH ortholog but rather a new class of divergent KAS I/II enzymes to initiate the FAS pathway. When a P. aeruginosa cosmid library was used to rescue growth in a fabH downregulated strain of Escherichia coli, a single unannotated open reading frame, PA5174, complemented fabH depletion. While deletion of all four KASIII domain-encoding genes in the same P. aeruginosa strain resulted in a wild-type growth phenotype, deletion of PA5174 alone specifically attenuated growth due to a defect in de novo FAS. Siderophore secretion and quorum-sensing signaling, particularly in the rhl and Pseudomonas quinolone signal (PQS) systems, was significantly muted in the absence of PA5174. The defect could be repaired by intergeneric complementation with E. coli fabH. Characterization of recombinant PA5174 confirmed a preference for short-chain acyl coenzyme A (acyl-CoA) substrates, supporting the identification of PA5174 as the predominant enzyme catalyzing the condensation of acetyl coenzyme A with malonyl-ACP in P. aeruginosa. The identification of the functional role for PA5174 in FAS defines the new FabY class of β-ketoacyl synthase KASI/II domain condensation enzymes. PMID:22753059

  19. The Mechanisms Underlying the Hypolipidaemic Effects of Grifola frondosa in the Liver of Rats

    PubMed Central

    Ding, Yinrun; Xiao, Chun; Wu, Qingping; Xie, Yizhen; Li, Xiangmin; Hu, Huiping; Li, Liangqiu

    2016-01-01

    The present study investigated the hypolipidaemic effects of Grifola frondosa and its regulation mechanism involved in lipid metabolism in liver of rats fed a high-cholesterol diet. The body weights and serum lipid levels of control rats, of hyperlipidaemic rats, and of hyperlipidaemic rats treated with oral G. frondosa were determined. mRNA expression and concentration of key lipid metabolism enzymes were investigated. Serum cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels were markedly decreased in hyperlipidaemic rats treated with G. frondosa compared with untreated hyperlipidaemic rats. mRNA expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), acyl-coenzyme A: cholesterol acyltransferase (ACAT2), apolipoprotein B (ApoB), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC1) were significantly down-regulated, while expression of cholesterol 7-alpha-hydroxylase (CYP7A1) was significantly up-regulated in the livers of treated rats compared with untreated hyperlipidaemic rats. The concentrations of these enzymes also paralleled the observed changes in mRNA expression. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) were used to identify 20 proteins differentially expressed in livers of rats treated with G. frondosa compared with untreated hyperlipidemic rats. Of these 20 proteins, seven proteins were down-regulated, and 13 proteins were up-regulated. These findings indicate that the hypolipidaemic effects of G. frondosa reflected its modulation of key enzymes involved in cholesterol and triacylglycerol biosynthesis, absorption, and catabolic pathways. G. frondosa may exert anti-atherosclerotic effects by inhibiting LDL oxidation through down-regulation and up-regulating proteins expression in the liver of rats. Therefore, G. frondosa may produce both hypolipidaemic and anti-atherosclerotic effects, and potentially

  20. Reduced capacity for fatty acid oxidation in rats with inherited susceptibility to diet-induced obesity.

    PubMed

    Ji, Hong; Friedman, Mark I

    2007-08-01

    High-fat, energy-dense diets promote weight gain and obesity in humans and other animals, but the mechanisms underlying such diet-induced obesity remain elusive. To determine whether a reduced capacity to oxidize fat is involved in the etiology of diet-induced obesity, we examined different measures of fatty acid oxidation in rats selectively bred for susceptibility (DIO) or resistance (DR) to dietary obesity before and after they were fed a high-fat diet and became obese. DIO rats eating a low-fat diet oxidized less dietary fatty acid in vivo and had lower levels of plasma ketone bodies during fasting compared with DR rats. Lean DIO rats fed a low-fat diet showed reduced liver messenger RNA expression of CD36, which transports fatty acids across cell membranes, and long-chain acyl-coenzyme A dehydrogenase (ACADL), which catalyzes the first step in the mitochondrial beta-oxidation of fatty acids. The deficit in CD36 and ACADL messenger RNA expression was also seen in obese DIO rats that had been eating a high-fat diet and, in addition, was accompanied by reduced expression of liver carnitine palmitoyl transferase I, the enzyme that mediates transport of long-chain fatty acids into mitochondria. No differences were found in the expression of liver enzymes involved in fat synthesis; however, in muscle, DIO rats fed the low-fat, but not high-fat, diet showed greater expression of diacylglycerol O-acyltransferase 1 and lipoprotein lipase than did DR rats. Expression of muscle enzymes involved in fatty acid oxidation was similar in the 2 groups. These findings provide a metabolic mechanism for the development of diet-induced obesity and thus suggest potential targets for intervention strategies to treat or prevent it. PMID:17618960

  1. Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae.

    PubMed Central

    Macreadie, I G; Novitski, C E; Maxwell, R J; John, U; Ooi, B G; McMullen, G L; Lukins, H B; Linnane, A W; Nagley, P

    1983-01-01

    A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein. PMID:6223276

  2. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1.

    PubMed Central

    Mohammadi, M; Honegger, A M; Rotin, D; Fischer, R; Bellot, F; Li, W; Dionne, C A; Jaye, M; Rubinstein, M; Schlessinger, J

    1991-01-01

    Phospholipase C-gamma (PLC-gamma) is a substrate of the fibroblast growth factor receptor (FGFR; encoded by the flg gene) and other receptors with tyrosine kinase activity. It has been demonstrated that the src homology region 2 (SH2 domain) of PLC-gamma and of other signalling molecules such as GTPase-activating protein and phosphatidylinositol 3-kinase-associated p85 direct their binding toward tyrosine-autophosphorylated regions of the epidermal growth factor or platelet-derived growth factor receptor. In this report, we describe the identification of Tyr-766 as an autophosphorylation site of flg-encoded FGFR by direct sequencing of a tyrosine-phosphorylated tryptic peptide isolated from the cytoplasmic domain of FGFR expressed in Escherichia coli. The same phosphopeptide was found in wild-type FGFR phosphorylated either in vitro or in living cells. Like other growth factor receptors, tyrosine-phosphorylated wild-type FGFR or its cytoplasmic domain becomes associated with intact PLC-gamma or with a fusion protein containing the SH2 domain of PLC-gamma. To delineate the site of association, we have examined the capacity of a 28-amino-acid tryptic peptide containing phosphorylated Tyr-766 to bind to various constructs containing SH2 and other domains of PLC-gamma. It is demonstrated that the tyrosine-phosphorylated peptide binds specifically to the SH2 domain but not to the SH3 domain or other regions of PLC-gamma. Hence, Tyr-766 and its flanking sequences represent a major binding site in FGFR for PLC-gamma. Alignment of the amino acid sequences surrounding Tyr-766 with corresponding regions of other FGFRs revealed conserved tyrosine residues in all known members of the FGFR family. We propose that homologous tyrosine-phosphorylated regions in other FGFRs also function as binding sites for PLC-gamma and therefore are involved in coupling to phosphatidylinositol breakdown. Images PMID:1656221

  3. An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

    PubMed Central

    Pos, Wouter; Crawley, James T. B.; Fijnheer, Rob; Voorberg, Jan; Lane, David A.

    2010-01-01

    In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (kcat/Km) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF. PMID:20032502

  4. Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull-down assay.

    PubMed

    Zhou, Zishan; Wang, Zeyu; Liu, Yuxiao; Liang, Gemei; Shu, Changlong; Song, Fuping; Zhou, Xueping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

    2016-08-01

    Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera. PMID:27037552

  5. Association of ventral striatum monoamine oxidase-A binding and functional connectivity in antisocial personality disorder with high impulsivity: A positron emission tomography and functional magnetic resonance imaging study.

    PubMed

    Kolla, Nathan J; Dunlop, Katharine; Downar, Jonathan; Links, Paul; Bagby, R Michael; Wilson, Alan A; Houle, Sylvain; Rasquinha, Fawn; Simpson, Alexander I; Meyer, Jeffrey H

    2016-04-01

    Impulsivity is a core feature of antisocial personality disorder (ASPD) associated with abnormal brain function and neurochemical alterations. The ventral striatum (VS) is a key region of the neural circuitry mediating impulsive behavior, and low monoamine oxidase-A (MAO-A) level in the VS has shown a specific relationship to the impulsivity of ASPD. Because it is currently unknown whether phenotypic MAO-A markers can influence brain function in ASPD, we investigated VS MAO-A level and the functional connectivity (FC) of two seed regions, superior and inferior VS (VSs, VSi). Nineteen impulsive ASPD males underwent [(11)C] harmine positron emission tomography scanning to measure VS MAO-A VT, an index of MAO-A density, and resting-state functional magnetic resonance imaging that assessed the FC of bilateral seed regions in the VSi and VSs. Subjects also completed self-report impulsivity measures. Results revealed functional coupling of the VSs with bilateral dorsomedial prefrontal cortex (DMPFC) that was correlated with VS MAO-A VT (r=0.47, p=0.04), and functional coupling of the VSi with right hippocampus that was anti-correlated with VS MAO-A VT (r=-0.55, p=0.01). Additionally, VSs-DMPFC FC was negatively correlated with NEO Personality Inventory-Revised impulsivity (r=-0.49, p=0.03), as was VSi-hippocampus FC with Barratt Impulsiveness Scale-11 motor impulsiveness (r=-0.50, p=0.03). These preliminary results highlight an association of VS MAO-A level with the FC of striatal regions linked to impulsive behavior in ASPD and suggest that phenotype-based brain markers of ASPD have relevance to understanding brain function. PMID:26908392

  6. Nuclear inclusions mimicking poly(A)-binding protein nuclear 1 inclusions in a case of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia with a novel mutation in the valosin-containing protein gene.

    PubMed

    Matsubara, Shiro; Shimizu, Toshio; Komori, Takashi; Mori-Yoshimura, Madoka; Minami, Narihiro; Hayashi, Yukiko K

    2016-07-01

    A middle-aged Japanese man presented with slowly progressive asymmetric weakness of legs and arm but had neither ptosis nor dysphagia. He had a family history of similar condition suggestive of autosomal dominant inheritance. A muscle biopsy showed mixture of neurogenic atrophy and myopathy with rimmed vacuoles. Furthermore we found intranuclear inclusions that had a fine structure mimicking that of inclusions reported in oculopharyngeal muscular dystrophy (OPMD). Immunohistochemical staining for polyadenylate-binding nuclear protein 1, which is identified within the nuclear inclusions of OPMD, demonstrated nuclear positivity in this case. However, OPMD was thought unlikely based on the clinical features and results of genetic analyses. Instead, a novel mutation in valosin-containing protein, c.376A>T (p.Ile126Phe), was revealed. A diagnosis of inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia was made. This is the first report of polyadenylate-binding nuclear protein 1-positive nuclear inclusions in the muscle of this condition. PMID:27209344

  7. The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation

    SciTech Connect

    LeNoue-Newton, Michele; Watkins, Guy R.; Zou, Ping; Germane, Katherine L.; McCorvey, Lisa R.; Wadzinski, Brian E.; Spiller, Benjamin W.

    2012-04-30

    Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: (1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and (2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

  8. Distribution of serotonin 5-HT1A-binding sites in the brainstem and the hypothalamus, and their roles in 5-HT-induced sleep and ingestive behaviors in rock pigeons (Columba livia).

    PubMed

    dos Santos, Tiago Souza; Krüger, Jéssica; Melleu, Fernando Falkenburger; Herold, Christina; Zilles, Karl; Poli, Anicleto; Güntürkün, Onur; Marino-Neto, José

    2015-12-15

    Serotonin 1A receptors (5-HT1ARs), which are widely distributed in the mammalian brain, participate in cognitive and emotional functions. In birds, 5-HT1ARs are expressed in prosencephalic areas involved in visual and cognitive functions. Diverse evidence supports 5-HT1AR-mediated 5-HT-induced ingestive and sleep behaviors in birds. Here, we describe the distribution of 5-HT1ARs in the hypothalamus and brainstem of birds, analyze their potential roles in sleep and ingestive behaviors, and attempt to determine the involvement of auto-/hetero-5-HT1ARs in these behaviors. In 6 pigeons, the anatomical distribution of [(3)H]8-OH-DPAT binding in the rostral brainstem and hypothalamus was examined. Ingestive/sleep behaviors were recorded (1h) in 16 pigeons pretreated with MM77 (a heterosynaptic 5-HT1AR antagonist; 23 or 69 nmol) for 20 min, followed by intracerebroventricular ICV injection of 5-HT (N:8; 150 nmol), 8-OH-DPAT (DPAT, a 5-HT1A,7R agonist, 30 nmol N:8) or vehicle. 5-HT- and DPAT-induced sleep and ingestive behaviors, brainstem 5-HT neuronal density and brain 5-HT content were examined in 12 pigeons, pretreated by ICV with the 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) or vehicle (N:6/group). The distribution of brainstem and diencephalic c-Fos immunoreactivity after ICV injection of 5-HT, DPAT or vehicle (N:5/group) into birds provided with or denied access to water is also described. 5-HT1ARs are concentrated in the brainstem 5-HTergic areas and throughout the periventricular hypothalamus, preoptic nuclei and circumventricular organs. 5-HT and DPAT produced a complex c-Fos expression pattern in the 5-HT1AR-enriched preoptic hypothalamus and the circumventricular organs, which are related to drinking and sleep regulation, but modestly affected c-Fos expression in 5-HTergic neurons. The 5-HT-induced ingestivebehaviors and the 5-HT- and DPAT-induced sleep behaviors were reduced by MM77 pretreatment. 5,7-DHT increased sleep per se, decreased tryptophan hydroxylase expression in the raphe nuclei and decreased prosencephalic 5-HT release but failed to affect 5-HT- or DPAT-induced drinking or sleep behavior. 5-HT- and DPAT-induced ingestive and sleep behaviors in pigeons appear to be mediated by heterosynaptic and/or non-somatodendri