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Sample records for acyltransferases

  1. Glycerophosphate/Acylglycerophosphate Acyltransferases

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    Atsushi Yamashita

    2014-11-01

    Full Text Available Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT and acyl-CoA: 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT are involved in the de novo synthesis of triacylglycerol (TAG and glycerophospholipids. Many enzymes belonging to the GPAT/AGPAT family have recently been identified and their physiological or pathophysiological roles have been proposed. The roles of GPAT/AGPAT in the synthesis of TAG and obesity-related diseases were revealed through the identification of causative genes of these diseases or analyses of genetically manipulated animals. Recent studies have suggested that some isoforms of GPAT/AGPAT family enzymes are involved in the fatty acid remodeling of phospholipids. The enzymology of GPAT/AGPAT and their physiological/ pathological roles in the metabolism of glycerolipids have been described and discussed in this review.

  2. Separation of 1-acylglycerolphosphate acyltransferase and 1-acylglycerolphosphorylcholine acyltransferase of rat liver microsomes.

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    Yamashita, S; Nakaya, N; Miki, Y; Numa, S

    1975-01-01

    1-Acylglycerolphosphate acyltransferase (Ec 2.3.1-) and 1-acylglycerolphosphorylcholine acyltransferase (EC 2.3.1.23) of rat liver microsomes were separated from each other. The separation was achieved by sucrose density gradient centrifugation of the enzyme preparation that was obtained by solubilizing microsomes with a nonionic detergent, Triton X-100, and subjecting the solubilized microsomes to molecular-sieve chromatography. The two acyltransferases are distinguishable from each other also with respect to their stabilities to heat and to Triton X-100. Hence, it is concluded that these acyltransferases are distinct enzymes. These results, together with our previous finding that glycerolphosphate acyltransferase is also a separate enzyme, demonstrate the presence of distinct acyltransferases responsible for the acylation of the different acyl acceptors. Furthermore, the acyl-donor specificities of these acyltransferases provide the enzymatic basis for the nonrandom distribution of fatty acids in naturally occurring glycerolipids. PMID:1054842

  3. Genomics of the human carnitine acyltransferase genes

    NARCIS (Netherlands)

    van der Leij, FR; Huijkman, NCA; Boomsma, C; Kuipers, JRG; Bartelds, B

    2000-01-01

    Five genes in the human genome are known to encode different active forms of related carnitine acyltransferases: CPT1A for liver-type carnitine palmitoyltransferase I, CPT1B for muscle-type carnitine palmitoyltransferase I, CPT2 for carnitine palmitoyltransferase II, CROT for carnitine octanoyltrans

  4. Acyl-coenzyme A:cholesterol acyltransferases

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    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...

  5. Structural Basis for the Acyltransferase Activity of Lecithin: Retinol Acyltransferase-like Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof (Case Western); (Columbia)

    2012-10-10

    Lecithin:retinol acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to lecithin:retinol acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.

  6. Soybean oil biosynthesis: role of diacylglycerol acyltransferases.

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    Li, Runzhi; Hatanaka, Tomoko; Yu, Keshun; Wu, Yongmei; Fukushige, Hirotada; Hildebrand, David

    2013-03-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to form seed oil triacylglycerol (TAG). To understand the features of genes encoding soybean (Glycine max) DGATs and possible roles in soybean seed oil synthesis and accumulation, two full-length cDNAs encoding type 1 diacylglycerol acyltransferases (GmDGAT1A and GmDGAT1B) were cloned from developing soybean seeds. These coding sequences share identities of 94 % and 95 % in protein and DNA sequences. The genomic architectures of GmDGAT1A and GmDGAT1B both contain 15 introns and 16 exons. Differences in the lengths of the first exon and most of the introns were found between GmDGAT1A and GmDGAT1B genomic sequences. Furthermore, detailed in silico analysis revealed a third predicted DGAT1, GmDGAT1C. GmDGAT1A and GmDGAT1B were found to have similar activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-diacylglycerol were preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. Both transcripts are much more abundant in developing seeds than in other tissues including leaves, stem, roots, and flowers. Both soybean DGAT1A and DGAT1B are highly expressed at developing seed stages of maximal TAG accumulation with DGAT1B showing highest expression at somewhat later stages than DGAT1A. DGAT1A and DGAT1B show expression profiles consistent with important roles in soybean seed oil biosynthesis and accumulation.

  7. Allostery and conformational dynamics in cAMP-binding acyltransferases.

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    Podobnik, Marjetka; Siddiqui, Nida; Rebolj, Katja; Nambi, Subhalaxmi; Merzel, Franci; Visweswariah, Sandhya S

    2014-06-06

    Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.

  8. Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.

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    Petrova, Elissaveta; Rios-Esteves, Jessica; Ouerfelli, Ouathek; Glickman, J Fraser; Resh, Marilyn D

    2013-04-01

    Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a new target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling.

  9. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    Science.gov (United States)

    2014-08-01

    regulation of synaptic and neuronal functions.17 A point mutation in DHHC21 was identified in the depilated (dep) mouse mutant, resulting in hair follicle ...and hair follicle differentiation. PLoS Genet. 5, e1000748. (19) Mansilla, F., Birkenkamp-Demtroder, K., Kruhoffer, M., Sorensen, F. B., Andersen, C...AWARD NUMBER: W81XWH-13-1-0203 TITLE: Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma PRINCIPAL INVESTIGATOR: Xu Wu

  10. Inhibitors of Hedgehog Acyltransferase Block Sonic Hedgehog Signaling

    OpenAIRE

    Petrova, Elissaveta; Rios-Esteves, Jessica; Ouerfelli, Ouathek; Glickman, J. Fraser; Resh, Marilyn D.

    2013-01-01

    Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a novel target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling.

  11. Lysophospholipid acyltransferases and eicosanoid biosynthesis in zebrafish myeloid cells.

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    Zarini, Simona; Hankin, Joseph A; Murphy, Robert C; Gijón, Miguel A

    2014-10-01

    Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species.

  12. Alterations in plasma lecithin : cholesterol acyltransferase and myeloperoxidase in acute myocardial infarction: Implications for cardiac outcome

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; Tietge, Uwe J. F.; Kwakernaak, Arjan J.; Dikkeschei, Bert D.; Perton, Frank; Tio, Rene A.

    2014-01-01

    Background: The cholesterol esterifying enzyme, lecithin: cholesterol acyltransferase (LCAT), plays a key role in HDL maturation and remodeling. Myeloperoxidase (MPO) may compromise LCAT enzymatic activity. We tested the extent to which plasma LCAT activity is altered in acute myocardial infarction

  13. A look at diacylglycerol acyltransferases (DGATs) in algae.

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    Chen, Jit Ern; Smith, Alison G

    2012-11-30

    Triacylglycerols (TAGs) from algae are considered to be a potentially viable source of biodiesel and thereby renewable energy, but at the moment very little is known about the biosynthetic pathway in these organisms. Here we compare what is currently known in eukaryotic algal species, in particular the characteristics of algal diacylglycerol acyltransferase (DGAT), the last enzyme of de novo TAG biosynthesis. Several studies in plants and mammals have shown that there are two DGAT isoforms, DGAT1 and DGAT2, which catalyse the same reaction but have no clear sequence similarities. Instead, they have differences in functionality and spatial and temporal expression patterns. Bioinformatic searches of sequenced algal genomes reveal that most algae have multiple copies of putative DGAT2s, whereas other eukaryotes have single genes. Investigating whether these putative isoforms are indeed functional and whether they confer significantly different phenotypes to algal cells will be vital for future efforts to genetically modify algae for biofuel production.

  14. Polyketide proofreading by an acyltransferase-like enzyme.

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    Jensen, Katja; Niederkrüger, Holger; Zimmermann, Katrin; Vagstad, Anna L; Moldenhauer, Jana; Brendel, Nicole; Frank, Sarah; Pöplau, Petra; Kohlhaas, Christoph; Townsend, Craig A; Oldiges, Marco; Hertweck, Christian; Piel, Jörn

    2012-03-23

    Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for polyketide titer improvement and biosynthetic investigations.

  15. A Vernonia Diacylglycerol Acyltransferase Can Increase Renewable Oil Production.

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    Hatanaka, Tomoko; Serson, William; Li, Runzhi; Armstrong, Paul; Yu, Keshun; Pfeiffer, Todd; Li, Xi-Le; Hildebrand, David

    2016-09-28

    Increasing the production of plant oils such as soybean oil as a renewable resource for food and fuel is valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. This study shows that by manipulating a highly active acyl-CoA:diacylglycerol acyltransferase (DGAT) the hydrocarbon flux to oil in oilseeds can be increased without reducing the protein component. Compared to other plant DGATs, a DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in yeast, insect cells, and soybean. Soybean lines expressing VgDGAT1A show a 4% increase in oil content without reductions in seed protein contents or yield per unit land area. Incorporation of this trait into 50% of soybeans worldwide could result in an increase of 850 million kg oil/year without new land use or inputs and be worth ∼U.S.$1 billion/year at 2012 production and market prices.

  16. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer.

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    Mansilla, Francisco; da Costa, Kerry-Ann; Wang, Shuli; Kruhøffer, Mogens; Lewin, Tal M; Orntoft, Torben F; Coleman, Rosalind A; Birkenkamp-Demtröder, Karin

    2009-01-01

    The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

  17. BAHD or SCPL acyltransferase? What a dilemma for acylation in the world of plant phenolic compounds.

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    Bontpart, Thibaut; Cheynier, Véronique; Ageorges, Agnès; Terrier, Nancy

    2015-11-01

    Phenolic compounds are secondary metabolites involved in several plant growth and development processes, including resistance to biotic and abiotic stresses. The biosynthetic pathways leading to the vast diversity of plant phenolic products often include an acylation step, with phenolic compounds being the donor or acceptor molecules. To date, two acyltransferase families using phenolic compounds as acceptor or donor molecules have been described, with each using a different 'energy-rich' acyl donor. BAHD-acyltransferases, named after the first four biochemically characterized enzymes of the group, use acyl-CoA thioesters as donor molecules, whereas SCPL (Serine CarboxyPeptidase Like)-acyltransferases use 1-O-β-glucose esters. Here, common and divergent specifications found in the literature for both enzyme families were analyzed to answer the following questions. Are both acyltransferases involved in the synthesis of the same molecule (or same group of molecules)? Are both acyltransferases recruited in the same plant? How does the subcellular localization of these enzymes impact metabolite trafficking in plant cells?

  18. High-level expression of Candida parapsilosis lipase/acyltransferase in Pichia pastoris.

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    Brunel, Laetitia; Neugnot, Virginie; Landucci, Laure; Boze, Hélène; Moulin, Guy; Bigey, Frédéric; Dubreucq, Eric

    2004-07-01

    Candida parapsilosis has been previously shown to produce a lipase/acyltransferase (EC 3.1.1.3) that preferentially catalyses transfer reactions such as alcoholysis over hydrolysis in the presence of suitable nucleophiles other than water, even in aqueous media (aw > 0.9 ). This enzyme has been shown to belong to a new family of lipases. The present work describes the cloning of the gene coding for this lipase/acyltransferase in the yeast Pichia pastoris and the heterologous high-level expression of the recombinant enzyme. The lipase/acyltransferase gene, in which the sequence encoding the signal peptide was replaced by that of the alpha-factor of Saccharomyces cerevisiae, was placed under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). A transformed P. pastoris clone, containing five copies of the lipase/acyltransferase gene, was selected for the production of recombinant enzyme. The fed-batch culture supernatant contained 5.8 gl(-1) (weighted) of almost pure recombinant lipase/acyltransferase displaying the same catalytic behavior as the original enzyme.

  19. Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

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    Klasson K Thomas

    2011-07-01

    Full Text Available Abstract Background Diacylglycerol acyltransferases (DGATs catalyze the final and rate-limiting step of triacylglycerol (TAG biosynthesis in eukaryotic organisms. Database search has identified at least 59 DGAT1 sequences from 48 organisms, but the expression of any DGAT1 as a full-length protein in E. coli had not been reported because DGAT1s are integral membrane proteins and difficult to express and purify. The objective of this study was to establish a procedure for expressing full-length DGAT1 in E. coli. Results An expression plasmid containing the open reading frame for tung tree (Vernicia fordii DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in E. coli BL21(DE3. Immunoblotting showed that the recombinant DGAT1 (rDGAT1 was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The rDGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized rDGAT1 was partially purified by Ni-NTA affinity chromatography. PreScission protease digestion confirmed the identity of rDGAT1 and showed extensive precipitation following Ni-NTA affinity purification. Conclusions This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system. The results suggest that recombinant DGAT1 is degraded extensively from the carboxyl terminus and associated with other proteins, lipids, and membranes.

  20. Identification of apolipoprotein N-acyltransferase (Lnt) in mycobacteria.

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    Tschumi, Andreas; Nai, Corrado; Auchli, Yolanda; Hunziker, Peter; Gehrig, Peter; Keller, Peter; Grau, Thomas; Sander, Peter

    2009-10-02

    Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.

  1. The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase.

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

    2005-02-01

    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.

  2. Overexpression of peanut diacylglycerol acyltransferase 2 in Escherichia coli.

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    Zhenying Peng

    Full Text Available Diacylglycerol acyltransferase (DGAT is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2 genes were cloned from the peanut cultivar 'Luhua 14' using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST fusion proteins in Escherichia coli Rosetta (DE3. Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a-GST, or AhDGAT2b-GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a-GST and AhDGAT2b-GST proteins increased the sizes of the host cells by 2.4-2.5 times that of the controls (post-IPTG induction. The total fatty acid (FA levels of the AhDGAT2a-GST and AhDGAT2a-GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for

  3. Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.

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    James R Petrie

    Full Text Available BACKGROUND: Monoacylglycerol acyltransferases (MGATs are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG pathway by acylating MAG to form diacylglycerol (DAG. Typical plant triacylglycerol (TAG biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT. Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.

  4. Two polyketide-synthase-associated acyltransferases are required for sulfolipid biosynthesis in Mycobacterium tuberculosis.

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    Bhatt, Kiranmai; Gurcha, Sudagar S; Bhatt, Apoorva; Besra, Gurdyal S; Jacobs, William R

    2007-02-01

    The methyl-branched fatty acyl components of sulfolipid-I (SL-I), a major glycolipid of the human pathogen Mycobacterium tuberculosis, are synthesized by the polyketide synthase Pks2. Rv3824c (papA1), located downstream of pks2, encodes a protein that belongs to a subfamily of acyltransferases associated with mycobacterial polyketide synthases [polyketide synthase-associated proteins (PAPs)]. The presence of a conserved acyltransferase motif (HX(3)DX(14)Y) suggested a role for PapA1 in acylation of sulfated trehalose to form SL-I. Targeted deletion of the H37Rv papA1 resulted in loss of SL-I, demonstrating its role in mycobacterial sulfolipid biosynthesis. Furthermore, SL-I synthesis was restored in the mutant strain following complementation with papA1, but not with mutant alleles of papA1 containing alterations of key residues in the acyltransferase motif, confirming that PapA1 was an acyltransferase. While other M. tuberculosis pks clusters are associated with a single PAP-encoding gene, it was demonstrated that another open reading frame, Rv3820c (papA2), located 5.8 kb downstream of papA1 is also an acyltransferase gene involved in SL-I biosynthesis: deletion of papA2 abolished SL-I production. The absence of any partially acylated intermediates in either null mutant indicated that both PapA1 and PapA2 were required for all acylation steps of SL-I assembly.

  5. Functional roles of three cutin biosynthetic acyltransferases in cytokinin responses and skotomorphogenesis.

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    Wu, Lei; Zhou, Zhao-Yang; Zhang, Chun-Guang; Chai, Juan; Zhou, Qin; Wang, Li; Hirnerová, Eva; Mrvková, Michaela; Novák, Ondřej; Guo, Guang-Qin

    2015-01-01

    Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

  6. Functional roles of three cutin biosynthetic acyltransferases in cytokinin responses and skotomorphogenesis.

    Directory of Open Access Journals (Sweden)

    Lei Wu

    Full Text Available Cytokinins (CKs regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1, whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr. GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase with diacylglycerol acyltransferase (DGAT activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8 double mutant [defective in glycerol-3-phosphate (G3P acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1, which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

  7. A new class of ghrelin O-acyltransferase inhibitors incorporating triazole-linked lipid mimetic groups.

    Science.gov (United States)

    Zhao, Feifei; Darling, Joseph E; Gibbs, Richard A; Hougland, James L

    2015-07-15

    Inhibitors of ghrelin O-acyltransferase (GOAT) have untapped potential as therapeutics targeting obesity and diabetes. We report the first examples of GOAT inhibitors incorporating a triazole linkage as a biostable isosteric replacement for the ester bond in ghrelin and amide bonds in previously reported GOAT inhibitors. These triazole-containing inhibitors exhibit sub-micromolar inhibition of the human isoform of GOAT (hGOAT), and provide a foundation for rapid future chemical diversification and optimization of hGOAT inhibitors.

  8. Involvement of the Phospholipid Sterol Acyltransferase1 in Plant Sterol Homeostasis and Leaf Senescence1[W

    Science.gov (United States)

    Bouvier-Navé, Pierrette; Berna, Anne; Noiriel, Alexandre; Compagnon, Vincent; Carlsson, Anders S.; Banas, Antoni; Stymne, Sten; Schaller, Hubert

    2010-01-01

    Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging. PMID:19923239

  9. The rv1184c locus encodes Chp2, an acyltransferase in Mycobacterium tuberculosis polyacyltrehalose lipid biosynthesis.

    Science.gov (United States)

    Touchette, Megan H; Holsclaw, Cynthia M; Previti, Mary L; Solomon, Viven C; Leary, Julie A; Bertozzi, Carolyn R; Seeliger, Jessica C

    2015-01-01

    Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.

  10. Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers

    OpenAIRE

    Li, Yonghua; Beisson, Fred; Koo, Abraham J. K.; Molina, Isabel; Pollard, Mike; Ohlrogge, John

    2007-01-01

    Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases es...

  11. Genome-wide identification and analysis of membrane-bound O-acyltransferase (MBOAT) gene family in plants.

    Science.gov (United States)

    Wang, Peng; Wang, Zhunian; Dou, Yongchao; Zhang, Xiaoxiao; Wang, Maoyuan; Tian, Xinmin

    2013-11-01

    Membrane bound O-acyl transferase (MBOAT) family is composed of gene members encoding a variety of acyltransferase enzymes, which play important roles in plant acyl lipid metabolism. Here, we present the first genome-enabled identification and analysis of MBOAT gene models in plants. In total, we identified 136 plant MBOAT sequences from 14 plant species with complete genomes. Phylogenetic relationship analyses suggested the plant MBOAT gene models fell into four major groups, two of which likely encode enzymes of diacylglycerol acyltransferase 1 (DGAT1) and lysophospholipid acyltransferase (LPLAT), respectively, with one-three copies of paralogs present in each of the most plant species. A group of gene sequences, which are homologous to Saccharomyces cerevisiae glycerol uptake proteins (GUP), was identified in plants; copy numbers were conserved, with only one copy represented in each of the most plant species; analyses showed that residues essential for acyltransferases were more prone to be conserved than vertebrate orthologs. Among four groups, one was inferred to emerge in land plants and experience a rapid expansion in genomes of angiosperms, which suggested their important roles in adaptation of plants in lands. Sequence and phylogeny analyses indicated that genes in all four groups encode enzymes with acyltransferases. Comprehensive sequence identification of MBOAT family members and investigation into classification provide a complete picture of the MBOAT gene family in plants, and could shed light into enzymatic functions of different MBOAT genes in plants.

  12. Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase.

    Science.gov (United States)

    Rani, Sapa Hima; Krishna, T H Anantha; Saha, Saikat; Negi, Arvind Singh; Rajasekharan, Ram

    2010-12-03

    A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

  13. Identification of acyltransferases required for cutin biosynthesis and production of cutin with suberin-like monomers.

    Science.gov (United States)

    Li, Yonghua; Beisson, Fred; Koo, Abraham J K; Molina, Isabel; Pollard, Mike; Ohlrogge, John

    2007-11-13

    Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases essential for cutin biosynthesis, glycerol-3-phosphate acyltransferase (GPAT) 4 and GPAT8. Double knockouts gpat4/gpat8 were strongly reduced in cutin and were less resistant to desiccation and to infection by the fungus Alternaria brassicicola. They also showed striking defects in stomata structure including a lack of cuticular ledges between guard cells, highlighting the importance of cutin in stomatal biology. Overexpression of GPAT4 or GPAT8 in Arabidopsis increased the content of C16 and C18 cutin monomers in leaves and stems by 80%. In order to modify cutin composition, the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis, were overexpressed. When both enzymes were overexpressed together the epidermal polyesters accumulated new C20 and C22 omega-hydroxyacids and alpha,omega-diacids typical of suberin, and the fine structure and water-barrier function of the cuticle were altered. These results identify GPATs as partners of fatty acyl oxidases in lipid polyester synthesis and indicate that their cooverexpression provides a strategy to probe the role of cutin composition and quantity in the function of plant cuticles.

  14. Discovery of a novel series of benzimidazole derivatives as diacylglycerol acyltransferase inhibitors.

    Science.gov (United States)

    Lee, Kyeong; Goo, Ja-Il; Jung, Hwa Young; Kim, Minkyoung; Boovanahalli, Shanthaveerappa K; Park, Hye Ran; Kim, Mun-Ock; Kim, Dong-Hyun; Lee, Hyun Sun; Choi, Yongseok

    2012-12-15

    A novel series of benzimidazole derivatives was prepared and evaluated for their diacylglycerol acyltransferase (DGAT) inhibitory activity using microsome from rat liver. Among the newly synthesized compounds, furfurylamine containing benzimidazole carboxamide 10j showed the most potent DGAT inhibitory effect (IC(50)=4.4 μM) and inhibited triglyceride formation in HepG2 cells. Furthermore, compound 10j reduced body weight gain of Institute of Cancer Research mice on a high-fat diet and decreased levels of total triglyceride, total cholesterol, and LDL-cholesterol in the blood accompanied with a significant increase in HDL-cholesterol level.

  15. Diagnosis in bile acid-CoA: Amino acid N-acyltransferase deficiency

    Institute of Scientific and Technical Information of China (English)

    Nedim Had(z)i(c); Laura N Bull; Peter T Clayton; AS Knisely

    2012-01-01

    Cholate-CoA ligase (CCL) and bile acid-CoA:amino acid N-acyltransferase (BAAT) sequentially mediate bile-acid amidation.Defects can cause intrahepatic cholestasis.Distinction has required gene sequencing.We assessed potential clinical utility of immunostaining of liver for CCL and BAAT.Using commercially available antibodies against BAAT and CCL,we immunostained liver from an infant with jaundice,deficiency of amidated bile acids,and transcription-terminating mutation in BAAT.CCL was normally expressed.BAAT expression was not detected.Immunostaining may facilitate diagnosis in bileacid amidation defects.

  16. Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology.

    Science.gov (United States)

    Liu, Qin; Siloto, Rodrigo M P; Lehner, Richard; Stone, Scot J; Weselake, Randall J

    2012-10-01

    Triacylglycerol (TG) is a storage lipid which serves as an energy reservoir and a source of signalling molecules and substrates for membrane biogenesis. TG is essential for many physiological processes and its metabolism is widely conserved in nature. Acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final step in the sn-glycerol-3-phosphate pathway leading to TG. DGAT activity resides mainly in two distinct membrane bound polypeptides, known as DGAT1 and DGAT2 which have been identified in numerous organisms. In addition, a few other enzymes also hold DGAT activity, including the DGAT-related acyl-CoA:monoacylglycerol acyltransferases (MGAT). Progress on understanding structure/function in DGATs has been limited by the lack of detailed three-dimensional structural information due to the hydrophobic properties of theses enzymes and difficulties associated with purification. This review examines several aspects of DGAT and MGAT genes and enzymes, including current knowledge on their gene structure, expression pattern, biochemical properties, membrane topology, functional motifs and subcellular localization. Recent progress in probing structural and functional aspects of DGAT1 and DGAT2, using a combination of molecular and biochemical techniques, is emphasized. Biotechnological applications involving DGAT enzymes ranging from obesity therapeutics to oilseed engineering are also discussed.

  17. Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids.

    Science.gov (United States)

    Akagi, Sosuke; Kono, Nozomu; Ariyama, Hiroyuki; Shindou, Hideo; Shimizu, Takao; Arai, Hiroyuki

    2016-05-01

    The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by dietary consumption of fatty acids such as saturated fatty acids and polyunsaturated fatty acids (PUFAs). Cells must adapt to changes in composition of membrane fatty acids by regulating lipid-metabolizing enzymes. In this study, we investigated how cells respond to loading with excess PUFAs, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. A lipidomics analysis revealed that dipalmitoylphosphatidylcholine (DPPC) was increased after the production of PUFA-containing phospholipids in cells loaded with PUFAs. An RNA interference screen of lipid-metabolizing enzymes revealed that lysophosphatidylcholine acyltransferase 1 (LPCAT1) was involved in the DPPC production. Moreover, LPCAT1 knockdown markedly enhanced the cytotoxicity induced by excess PUFAs. PUFA-induced cytotoxicity was dependent on caspase and unfolded protein response (UPR) sensor proteins inositol requiring 1α and protein kinase R-like endoplasmic reticulum kinase, suggesting that excess PUFAs trigger UPR-mediated apoptosis. In murine retina, in which PUFAs are highly enriched, DPPC was produced along with increase of PUFA-containing phospholipids. In LPCAT1 knockout mice, DPPC level was reduced and UPR was activated in the retina. Our results provide insight into understanding of the retinal degeneration seen in rd11 mice that lack LPCAT1.-Akagi, S., Kono, N., Ariyama, H., Shindou, H., Shimizu, T., Arai, H. Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids.

  18. The Pun1 gene for pungency in pepper encodes a putative acyltransferase.

    Science.gov (United States)

    Stewart, Charles; Kang, Byoung-Cheorl; Liu, Kede; Mazourek, Michael; Moore, Shanna L; Yoo, Eun Young; Kim, Byung-Dong; Paran, Ilan; Jahn, Molly M

    2005-06-01

    Pungency in Capsicum fruits is due to the accumulation of the alkaloid capsaicin and its analogs. The biosynthesis of capsaicin is restricted to the genus Capsicum and results from the acylation of an aromatic moiety, vanillylamine, by a branched-chain fatty acid. Many of the enzymes involved in capsaicin biosynthesis are not well characterized and the regulation of the pathway is not fully understood. Based on the current pathway model, candidate genes were identified in public databases and the literature, and genetically mapped. A published EST co-localized with the Pun1 locus which is required for the presence of capsaicinoids. This gene, AT3, has been isolated and its nucleotide sequence has been determined in an array of genotypes within the genus. AT3 showed significant similarity to acyltransferases in the BAHD superfamily. The recessive allele at this locus contains a deletion spanning the promoter and first exon of the predicted coding region in every non-pungent accession tested. Transcript and protein expression of AT3 was tissue-specific and developmentally regulated. Virus-induced gene silencing of AT3 resulted in a decrease in the accumulation of capsaicinoids, a phenotype consistent with pun1. In conclusion, gene mapping, allele sequence data, expression profile and silencing analysis collectively indicate that the Pun1 locus in pepper encodes a putative acyltransferase, and the pun1 allele, used in pepper breeding for nearly 50 000 years, results from a large deletion at this locus.

  19. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis.

    Science.gov (United States)

    Maisonneuve, Sylvie; Bessoule, Jean-Jacques; Lessire, René; Delseny, Michel; Roscoe, Thomas J

    2010-02-01

    In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.

  20. Involvement of an octose ketoreductase and two acyltransferases in the biosynthesis of paulomycins

    Science.gov (United States)

    Li, Jine; Wang, Min; Ding, Yong; Tang, Yue; Zhang, Zhiguo; Chen, Yihua

    2016-02-01

    C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4‧ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7‧-keto of PAU E (1) to give the C-4‧ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4‧ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7‧-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs.

  1. Saccharomyces cerevisiae lysophospholipid acyltransferase, Lpt1, requires Asp146 and Glu297 for catalysis.

    Science.gov (United States)

    Renauer, Paul; Nasiri, Nour; Oelkers, Peter

    2015-11-01

    The esterification of lysophospholipids contributes to phospholipid synthesis, remodeling, and scavenging. Acyl-CoA-dependent lysophospholipid acyltransferase activity with broad substrate use is mediated by Saccharomyces cerevisiae Lpt1p. We sought to identify Lpt1p active site amino acids besides the histidine conserved among homologs and repeatedly found to be required for catalysis. In vitro Lpt1p assays with amino acid modifying agents implicated aspartate, glutamate, and lysine as active site residues. Threonine and tyrosine were not ruled out. Aligning the primary structures of functionally characterized LPT1 homologs from fungi, plants, and animals identified 11 conserved aspartate, glutamate, lysine, threonine, and tyrosine residues. Site-directed mutagenesis of the respective codons showed that changing D146 and E297 abolished activity without abolishing protein expression. The mechanism of Lpt1p was further analyzed using monounsaturated acyl-CoA species with different double bond positions. Delta 6 species showed the highest catalytic efficiency. We propose that D146 and E297 act in conjunction with H382 as nucleophiles that attack the hydroxyl group in lysophospholipids in a general acid/base mechanism. This sequential mechanism provides a precedent for other members of the membrane bound O-acyltransferase family. Also, Lpt1p optimally orients acyl-CoA substrates with 7.5 Å between a double bond and the thioester bond.

  2. PapA3 is an acyltransferase required for polyacyltrehalose biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Hatzios, Stavroula K; Schelle, Michael W; Holsclaw, Cynthia M; Behrens, Christopher R; Botyanszki, Zsofia; Lin, Fiona L; Carlson, Brian L; Kumar, Pawan; Leary, Julie A; Bertozzi, Carolyn R

    2009-05-08

    Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.

  3. Diacylglycerol acyltransferase-1 inhibition enhances intestinal fatty acid oxidation and reduces energy intake in rats.

    Science.gov (United States)

    Schober, Gudrun; Arnold, Myrtha; Birtles, Susan; Buckett, Linda K; Pacheco-López, Gustavo; Turnbull, Andrew V; Langhans, Wolfgang; Mansouri, Abdelhak

    2013-05-01

    Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect.

  4. The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase.

    Science.gov (United States)

    Ma, Qingjun; Zhao, Xin; Nasser Eddine, Ali; Geerlof, Arie; Li, Xinping; Cronan, John E; Kaufmann, Stefan H E; Wilmanns, Matthias

    2006-06-06

    Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4'-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.

  5. Deficiency of acyl-CoA: Dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebro-hepato-renal) syndrome

    NARCIS (Netherlands)

    Bosch, H. van den; Schutgens, R.B.H.; Romeyn, G.J.; Wanders, R.J.A.; Schrakamp, G.; Heymans, H.S.A.

    1984-01-01

    We have recently reported on plasmalogen deficiency in tissues and fibroblasts from patients with Zellweger syndrome. In this paper we have analyzed the activity of the first enzyme in the pathway leading to plasmalogen biosynthesis, i.e. acyl-CoA: dihydroxyacetone phosphate acyltransferase in liver

  6. Homeostasis of Retinol in Lecithin:retinol Acyltransferase Gene Knockout Mice Fed a High Retinol Diet

    OpenAIRE

    Liu, Limin; Tang, Xiao-Han; Gudas, Lorraine J.

    2008-01-01

    We analyzed the retinoid levels and gene expression in various tissues after wild type (Wt) and lecithin:retinol acyltransferase knockout (LRAT−/−) mice were fed a high retinol diet (250 IU/gram). As compared to Wt, LRAT−/− mice exhibited a greater and faster increase in serum retinol concentration (mean±S.D., Wt, 1.3±0.2 µM to 1.5±0.3 µM in 48 hours, p>0.05; LRAT−/−, 1.3±0.2 µM to 2.2±0.3 µM in 48 hours, p

  7. Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum.

    Science.gov (United States)

    Lee, Seung Woong; Rho, Mun-Chual; Park, Hye Ran; Choi, Jung-Ho; Kang, Ji Yun; Lee, Jung Won; Kim, Koanhoi; Lee, Hyun Sun; Kim, Young Kook

    2006-12-27

    Pharmacological inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) has emerged as a potential therapy for the treatment of obesity and type 2 diabetes. Bioassay-guided isolation of CHCl3 extracts of the fruits of Piper longum and Piper nigum (Piperaceae), using an in vitro DGAT inhibitory assay, lead to isolation of a new alkamide named (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (2), together with four known alkamides: retrofractamide C (1), pipernonaline (3), piperrolein B (4), and dehydropipernonaline (5). Compounds 2-5 inhibited DGAT with IC50 values of 29.8 (2), 37.2 (3), 20.1 (4), and 21.2 (5) microM, respectively, but the IC50 value for 1 was more than 900 microM. This finding indicates that compounds possessing piperidine groups (2-5) can be potential DGAT inhibitors.

  8. Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

    DEFF Research Database (Denmark)

    Eudes, Aymerick; Mouille, Maxence; Robinson, David S.

    2016-01-01

    hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate......Background: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad...... syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals...

  9. Diacylglycerol acyltransferase-1 (DGAT1 inhibition perturbs postprandial gut hormone release.

    Directory of Open Access Journals (Sweden)

    Hua V Lin

    Full Text Available Diacylglycerol acyltransferase-1 (DGAT1 is a potential therapeutic target for treatment of obesity and related metabolic diseases. However, the degree of DGAT1 inhibition required for metabolic benefits is unclear. Here we show that partial DGAT1 deficiency in mice suppressed postprandial triglyceridemia, led to elevations in glucagon-like peptide-1 (GLP-1 and peptide YY (PYY only following meals with very high lipid content, and did not protect from diet-induced obesity. Maximal DGAT1 inhibition led to enhanced GLP-1 and PYY secretion following meals with physiologically relevant lipid content. Finally, combination of DGAT1 inhibition with dipeptidyl-peptidase-4 (DPP-4 inhibition led to further enhancements in active GLP-1 in mice and dogs. The current study suggests that targeting DGAT1 to enhance postprandial gut hormone secretion requires maximal inhibition, and suggests combination with DPP-4i as a potential strategy to develop DGAT1 inhibitors for treatment of metabolic diseases.

  10. The last step in cocaine biosynthesis is catalyzed by a BAHD acyltransferase.

    Science.gov (United States)

    Schmidt, Gregor Wolfgang; Jirschitzka, Jan; Porta, Tiffany; Reichelt, Michael; Luck, Katrin; Torre, José Carlos Pardo; Dolke, Franziska; Varesio, Emmanuel; Hopfgartner, Gérard; Gershenzon, Jonathan; D'Auria, John Charles

    2015-01-01

    The esterification of methylecgonine (2-carbomethoxy-3β-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.

  11. Activity and Crystal Structure of Arabidopsis thalianaUDP-N-Acetylglucosamine Acyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Sang Hoon; Chung, Hak Suk; Raetz, Christian R.H.; Garrett, Teresa A. (Vassar); (CUD- South Korea); (Duke)

    2012-08-31

    The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 {angstrom} resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an {alpha}-helical-rich C-terminus and characteristic N-terminal left-handed parallel {beta}-helix (L{beta}H). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the L{beta}H not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.

  12. Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea

    Indian Academy of Sciences (India)

    Si-Long Chen; Jia-Quan Huang; Lei Yong; Yue-Ting Zhang; Xiao-Ping Ren; Yu-Ning Chen; Hui-Fang Jiang; Li-Ying Yan; Yu-Rong Li; Bo-Shou Liao

    2012-12-01

    Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

  13. Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.

    Directory of Open Access Journals (Sweden)

    John A Buglino

    Full Text Available BACKGROUND: Sonic hedgehog (Shh is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat, a member of the membrane bound O-acyl transferase (MBOAT family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196-234 that is required for Hhat activity. In total, ten deletion mutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five point mutants retained stability but lost palmitoylation activity. We purified two point mutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent K(m and V(max for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. CONCLUSIONS/SIGNIFICANCE: This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases.

  14. Characterization of a Novel Intestinal Glycerol-3-phosphate Acyltransferase Pathway and Its Role in Lipid Homeostasis.

    Science.gov (United States)

    Khatun, Irani; Clark, Ronald W; Vera, Nicholas B; Kou, Kou; Erion, Derek M; Coskran, Timothy; Bobrowski, Walter F; Okerberg, Carlin; Goodwin, Bryan

    2016-02-01

    Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism.

  15. Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato.

    Science.gov (United States)

    Sui, Na; Li, Meng; Zhao, Shi-Jie; Li, Feng; Liang, Hui; Meng, Qing-Wei

    2007-10-01

    A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.

  16. Molecular characterization of a lysophosphatidylcholine acyltransferase gene belonging to the MBOAT family in Ricinus communis L.

    Science.gov (United States)

    Arroyo-Caro, José María; Chileh, Tarik; Alonso, Diego López; García-Maroto, Federico

    2013-07-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) catalyzes acylation of lysophosphatidylcholine (lysoPtdCho) to produce phosphatidylcholine (PtdCho), the main phospholipid in cellular membranes. This reaction is a key component of the acyl-editing process, involving recycling of the fatty acids (FA) mainly at the sn-2 position of PtdCho. Growing evidences indicate that the LPCAT reaction controls the direct entry of newly synthesized FA into PtdCho and, at least in some plant species, it has an important impact on the synthesis and composition of triacylglycerols. Here we describe the molecular characterization of the single LPCAT gene found in the genome of Ricinus communis (RcLPCAT) that is homologous to LPCAT genes of the MBOAT family previously described in Arabidopsis and Brassica. RcLPCAT is ubiquitously expressed in all organs of the castor plant. Biochemical properties have been studied by heterologous expression of RcLPCAT in the ale1 yeast mutant, defective in lysophospholipid acyltransferase activity. RcLPCAT preferentially acylates lysoPtdCho against other lysophospholipids (lysoPL) and does not discriminates the acyl chain in the acceptor, displaying a strong activity with alkyl lysoPL. Regarding the acyl-CoA donor, RcLPCAT uses monounsaturated fatty acid thioesters, such as oleoyl-CoA (18:1-CoA), as preferred donors, while it has a low activity with saturated fatty acids and shows a poor utilization of ricinoleoyl-CoA (18:1-OH-CoA). These characteristics are discussed in terms of a possible role of RcLPCAT in regulating the entry of FA into PtdCho and the exclusion from the membranes of the hydroxylated FA.

  17. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

    DEFF Research Database (Denmark)

    Benghezal, Mohammed; Roubaty, Carole; Veepuri, Vijayanath

    2007-01-01

    Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal an......-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids....

  18. The mycobacterial acyltransferase PapA5 is required for biosynthesis of cell wall-associated phenolic glycolipids.

    Science.gov (United States)

    Chavadi, Sivagami Sundaram; Onwueme, Kenolisa C; Edupuganti, Uthamaphani R; Jerome, Jeff; Chatterjee, Delphi; Soll, Clifford E; Quadri, Luis E N

    2012-05-01

    Phenolic glycolipids (PGLs) are non-covalently bound components of the outer membrane of many clinically relevant mycobacterial pathogens, and play important roles in pathogen biology. We report a mutational analysis that conclusively demonstrates that the conserved acyltransferase-encoding gene papA5 is essential for PGL production. In addition, we provide an in vitro acyltransferase activity analysis that establishes proof of principle for the competency of PapA5 to utilize diol-containing polyketide compounds of mycobacterial origin as acyl-acceptor substrates. Overall, the results reported herein are in line with a model in which PapA5 catalyses the acylation of diol-containing polyketides to form PGLs. These studies advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids and suggest that PapA5 might be an attractive target for exploring the development of antivirulence drugs.

  19. Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5.

    Science.gov (United States)

    Onwueme, Kenolisa C; Ferreras, Julian A; Buglino, John; Lima, Christopher D; Quadri, Luis E N

    2004-03-30

    Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acyltransferase [corrected]. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin.

  20. Purification and characterization of the acyltransferase involved in biosynthesis of the major mycobacterial cell envelope glycolipid--monoacylated phosphatidylinositol dimannoside.

    Science.gov (United States)

    Svetlíková, Zuzana; Baráth, Peter; Jackson, Mary; Korduláková, Jana; Mikušová, Katarína

    2014-08-01

    Phosphatidylinositol mannosides are essential structural components of the mycobacterial cell envelope. They are implicated in host-pathogen interactions during infection and serve as a basis for biosynthesis of other unique molecules with immunomodulatory properties - mycobacterial lipopolysaccharides lipoarabinomannan and lipomannan. Acyltransferase Rv2611 is involved in one of the initial steps in the assembly of these molecules in Mycobacterium tuberculosis - the attachment of an acyl group to position-6 of the 2-linked mannosyl residue of the phosphatidylinositol mannoside anchor. Although the function of this enzyme was annotated 10 years ago, it has never been completely biochemically characterized due to lack of the pure protein. We have successfully overexpressed and purified MSMEG_2934, the ortholog of Rv2611c from the non-pathogenic model organism Mycobacteriumsmegmatis mc(2)155 using mycobacterial pJAM2 expression system, which allowed confirmation of its in vitro acyltransferase activity, and establishment of its substrate specificity.

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

    Science.gov (United States)

    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.

  2. Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism.

    Science.gov (United States)

    Dovala, Dustin; Rath, Christopher M; Hu, Qijun; Sawyer, William S; Shia, Steven; Elling, Robert A; Knapp, Mark S; Metzger, Louis E

    2016-10-11

    Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and "reset" mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases' structure-mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.

  3. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  4. A molecular model for diacylglycerol acyltransferase from Mortierella ramanniana var. angulispora.

    Science.gov (United States)

    Mishra, Sanjay; Dwivedi, Surya Prakash; Dwivedi, Neeraja; Kumar, Ajay; Rawat, Anil; Kamisaka, Yasushi

    2009-06-28

    Acyl CoA diacylglycerol acyltransferase (DGAT, EC 2.3.120) is recognized as a key player of cellular diacylglycerol metabolism. It catalyzes the terminal, yet the committed step in triacylglycerol synthesis using diacylglycerol and fatty acyl CoA as substrates. The protein sequence of diacylglycerol acyltransferse (DGAT) Type 2B in Moretierella ramanniana var. angulispora (Protein_ID = AAK84180.1) was retrieved from GenBank. However, a structure is not yet available for this sequence. The 3D structure of DGAT Type 2B was modeled using a template structure (PDB ID: 1K30) obtained from Protein databank (PDB) identified by searching with position specific iterative BLAST (PSI-BLAST). The template (PDB ID: 1K30) describes the structure of DGAT from Cucurbita moschata. Modeling was performed using Modeller 9v2 and protein model is hence generated. The DGAT type 2B protein model was subsequently docked with six inhibitors (sphingosine; trifluoroperazine; phosphatidic acid; lysophospatidylserine; KCl; 1, 2-diolein) using AutoDock (a molecular docking program). The binding of inhibitors to the protein model of DGAT type 2B is discussed.

  5. Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured bacterial consortia.

    Science.gov (United States)

    Piel, Jörn; Hui, Dequan; Fusetani, Nobuhiro; Matsunaga, Shigeki

    2004-09-01

    Bacterial type I polyketide synthases (PKSs) produce a wide range of biomedically important secondary metabolites. These enzymes possess a modular structure that can be genetically re-engineered to yield novel drug candidates not found in nature. Recently, we have reported the putative pederin PKS from an uncultured bacterial symbiont of Paederus fuscipes beetles. It belongs to an architecturally unusual PKS group, the members of which contain iteratively acting acyltransferases that are not integrated into the PKS modules but are encoded by isolated genes. As these systems are rare, often contain additional unusual features and are of smaller size than regular PKSs, the development of a method for the targeted isolation of new group members would be of great interest. Here, we present a phylogenetic approach to identify these systems rapidly in highly complex metagenomic DNA samples. To demonstrate its practical value, we located two pederin-type PKS systems putatively involved in the biosynthesis of antitumour polyketides in the metagenomic DNA of beetles, sponges and their uncultivated bacterial symbionts.

  6. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.

    Science.gov (United States)

    Lanyon-Hogg, Thomas; Masumoto, Naoko; Bodakh, George; Konitsiotis, Antonio D; Thinon, Emmanuelle; Rodgers, Ursula R; Owens, Raymond J; Magee, Anthony I; Tate, Edward W

    2015-12-01

    Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

  7. Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis, in Arabidopsis thaliana.

    Science.gov (United States)

    Lu, Chaofu Lu; de Noyer, Shen Bayon; Hobbs, Douglas H; Kang, Jinling; Wen, Yancheng; Krachtus, Dieter; Hills, Matthew J

    2003-05-01

    Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene beta-glucuronidase (GUS) fused with DNA sequences flanking the DGAT1 coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1 gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.

  8. Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.

    Science.gov (United States)

    Liu, Limin; Gudas, Lorraine J

    2005-12-02

    Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.

  9. Current status of the research and development of diacylglycerol O-acyltransferase 1 (DGAT1) inhibitors.

    Science.gov (United States)

    DeVita, Robert J; Pinto, Shirly

    2013-12-27

    Diacylglycerol O-acyltransferase 1 (DGAT1) has recently become a highly interesting target for metabolic disorders as well as for hepatitis C virus (HCV). DGAT1 processes diacylglycerol to triglycerides in the final step of resynthesis for the absorption of fat across the intestine. Pharmaceutical companies have developed many novel inhibitors of DGAT1, several of which have reached the clinic. Proof of target engagement was achieved with the observation of reduced triglycerides upon treatment of humans with DGAT1 inhibitors; however, there were gastrointestinal adverse events such as nausea, diarrhea, and vomiting. These adverse events have been reported with multiple compounds and are possibly linked to the target because of the recent identification of a human cohort deficient in DGAT1. Clinical studies are continuing in a trial to treat patients with an orphan indication for familial chylomicronemia. The full potential of DGAT1 as a therapeutic target will need to overcome observed clinical adverse events, which are possibly mechanism based. The widespread use of DGAT1 inhibitors will ultimately depend upon a better understanding of how to improve the GI tolerability of these agents.

  10. A type 2 diacylglycerol acyltransferase accelerates the triacylglycerol biosynthesis in heterokont oleaginous microalga Nannochloropsis oceanica.

    Science.gov (United States)

    Li, Da-Wei; Cen, Shi-Ying; Liu, Yu-Hong; Balamurugan, Srinivasan; Zheng, Xin-Yan; Alimujiang, Adili; Yang, Wei-Dong; Liu, Jie-Sheng; Li, Hong-Ye

    2016-07-10

    Oleaginous microalgae have received a considerable attention as potential biofuel feedstock. However, lack of industry-suitable strain with lipid rich biomass limits its commercial applications. Targeted engineering of lipogenic pathways represents a promising strategy to enhance the efficacy of microalgal oil production. In this study, a type 2 diacylglycerol acyltransferase (DGAT), a rate-limiting enzyme in triacylglycerol (TAG) biosynthesis, was identified and overexpressed in heterokont oleaginous microalga Nannochloropsis oceanica for the first time. Overexpression of DGAT2 in Nannochloropsis increased the relative transcript abundance by 3.48-fold in engineered microalgae cells. TAG biosynthesis was subsequently accelerated by DGAT2 overexpression and neutral lipid content was significantly elevated by 69% in engineered microalgae. The fatty acid profile determined by GC-MS revealed that fatty acid composition was altered in engineered microalgae. Saturated fatty acids and polyunsaturated fatty acids were found to be increased whereas monounsaturated fatty acids content decreased. Furthermore, DGAT2 overexpression did not show negative impact on algal growth parameters. The present investigation showed that the identified DGAT2 would be a potential candidate for enhancing TAG biosynthesis and might facilitate the development of promising oleaginous strains with industrial potential.

  11. Lecithin-cholesterol acyltransferase in brain: Does oxidative stress influence the 24-hydroxycholesterol esterification?

    Science.gov (United States)

    La Marca, Valeria; Maresca, Bernardetta; Spagnuolo, Maria Stefania; Cigliano, Luisa; Dal Piaz, Fabrizio; Di Iorio, Giuseppe; Abrescia, Paolo

    2016-04-01

    24-Hydroxycholesterol (24OH-C) is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT) in the cerebrospinal fluid (CSF). We report here that the level of 24OH-C esters was lower in CSF of patients with amyotrophic lateral sclerosis than in healthy subjects (54% vs 68% of total 24OH-C, p=0.0005; n=8). Similarly, the level of 24OH-C esters in plasma was lower in patients than in controls (62% vs 77% of total 24OH-C; p=0.0076). The enzyme amount in CSF, as measured by densitometry of the protein band revealed by immunoblotting, was about 4-fold higher in patients than in controls (p=0.0085). As differences in the concentration of the LCAT stimulator Apolipoprotein E were not found, we hypothesized that the reduced 24OH-C esterification in CSF of patients might depend on oxidative stress. We actually found that oxidative stress reduced LCAT activity in vitro, and 24OH-C effectively stimulated the enzyme secretion from astrocytoma cells in culture. Enhanced LCAT secretion from astrocytes might represent an adaptive response to the increase of non-esterified 24OH-C percentage, aimed to avoid the accumulation of this neurotoxic compound. The low degree of 24OH-C esterification in CSF or plasma might reflect reduced activity of LCAT during neurodegeneration.

  12. Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis.

    Science.gov (United States)

    Yu, Keshun; Li, Runzhi; Hatanaka, Tomoko; Hildebrand, David

    2008-03-01

    Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil.

  13. AT3 (Acyltransferase Gene Isolated From Capsicum frutescens cv. Cakra Hijau

    Directory of Open Access Journals (Sweden)

    Mohamad Habibi

    2013-05-01

    Full Text Available Chili pepper is widely used and cultivated by Indonesian people. There are three species of chili pepper, i.e.: Capsicum annuum L., Capsicum frutescens L., and Capsicum violaceum HBK. Capsicum frutescens L. has a higher economic value due to its pungency and carotenoid content. C. frutescens has several cultivars, one of those is Capsicum frutescens cv. Cakra Hijau. This cultivar is resistant against pest and disease and has very high pungency. This special character of chili pepper is born by its secondary metabolic, Capsaicin. Moreover, capsaicin also serves as defense mechanism, antiarthritis, analgesic, and anticancer. This study aimed to isolate Acyltransferase (AT3 gene which encoding Capsaicin Synthase (CS enzyme. AT3 gene was isolated through PCR using forward primer 5’-ATG GCT TTT GCA TTA CCA TCA-3’ and reverse primer 5’-CCT TCA CAA TTA TTC GCC CA-3’. Data were analyzed using DNA Baser, BLAST, and ClustalX. This study has successfully isolated 404 bp fragments of AT3 gene. This fragments located at 1918-1434 bp referred to AT3 gene from Capsicum frutescens cv. Shuanla. Isolation of upstream and downstream fragments of AT3 gene from Capsicum frutescens cv. Cakra Hijau is undergoing.

  14. Acute sterol o-acyltransferase 2 (SOAT2 knockdown rapidly mobilizes hepatic cholesterol for fecal excretion.

    Directory of Open Access Journals (Sweden)

    Stephanie M Marshall

    Full Text Available The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE. We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2 increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD, the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼ 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.

  15. Glycerol-3-phosphate acyltransferase 4 gene is involved in mouse spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    Qingming Qiu; Gang Liu; Weina Li; Qiuwen Shi; Fuxi Zhu; Guangxiu Lu

    2009-01-01

    Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step of de novo triacylglycerol syn-thesis by converting glycerol-3-phosphate to lysopho-sphatidic acid (LPA). LPA is a mitogen that mediates multiple cellular processes including cell proliferation. Four GPAT isoforms have been cloned to date. GPAT4 is strongly expressed in the mouse testis. Reverse tran-scription-polymerase chain reaction (PCR), real-time PCR, and in situ hybridization (ISH) were used to analyze the GPAT4 expression and to localize the expressing cell types in the mouse testis during post-natal development. GPAT4 cDNA was inserted into pcDNA4/His to construct a recombinant vector, which was transfected into a mouse spermatogonial cell line (GC-lspg). GPAT4 was first expressed in mice at 2 weeks postnatally. Expression was abundant from the third week, plateaued at week 5-6 and then maintained at a high level in the adult. ISH revealed that GPAT4 gene was expressed abundantly in spermatocytes and around spermatids during meiosis but not in elongated spermatids during later spermiogenesis. GC-1spg cells showed a marked increase in proliferation after trans-fection with GPAT4; cell cycle analysis showed a decrease in the percentage of cells in the Go/G1 phase and an increase in the S phase. Thus, GPAT4 might play an important role in spermatogenesis, especially in mid-meiosis.

  16. Rapid ester biosynthesis screening reveals a high activity alcohol-O-acyltransferase (AATase) from tomato fruit.

    Science.gov (United States)

    Lin, Jyun-Liang; Zhu, Jie; Wheeldon, Ian

    2016-05-01

    Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.

  17. A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1.

    Science.gov (United States)

    Kalscheuer, Rainer; Steinbüchel, Alexander

    2003-03-07

    Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.

  18. Regulation of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in the rat adrenal gland.

    Science.gov (United States)

    Beins, D M; Vining, R; Balasubramaniam, S

    1982-03-15

    The activities of neutral cholesterol esterase and acyl-CoA : cholesterol acyltransferase in rat adrenal gland were measured at various time intervals over 24 h. The activity of cholesterol esterase displayed diurnal rhythm, with a major peak at the onset of darkness coinciding with the peak in the diurnal rhythm of plasma corticosterone concentration. The activity of acyl-CoA : cholesterol acyltransferase also exhibited a characteristic diurnal rhythm, with the minimum activity occurring 3 h after the onset of darkness. The profile of the rhythm exhibited by the activity of the esterifying enzyme was similar to the mirror image of the pattern of diurnal rhythm in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase. Microsomal non-esterified cholesterol showed a gradual decline with a significant decrease in concentration at the onset of darkness, thus suggesting that diurnal removal of cholesterol in the environment of the esterifying enzyme and hydroxymethylglutaryl-CoA reductase leads to such diurnal decrease or increase in the activities of these two enzymes. Acute administration of corticotropin led to a 3-fold increase in the activity of cholesterol esterase, a 50% decrease in the activity of acyl-CoA : cholesterol acyltransferase and a 2-fold increase in the activity of hydroxymethylglutaryl-CoA reductase. Corticotropin administration also resulted in a significant decrease in microsomal non-esterified cholesterol and increase in plasma corticosterone concentration. These observations suggest that corticotropin plays an important part in generating the diurnal rhythm in the activities of the three enzymes.

  19. Analysis of neutral lipid biosynthesis in Streptomyces avermitilis MA-4680 and characterization of an acyltransferase involved herein.

    Science.gov (United States)

    Kaddor, Chlud; Biermann, Karolin; Kalscheuer, Rainer; Steinbüchel, Alexander

    2009-08-01

    The physiology of lipid production in Streptomyces avermitilis MA-4680 with regard to the fatty acid composition of the accumulated lipids and their cellular distribution was analyzed. Cells were able to accumulate about ten to 30 lipid granules with diameters between 100 and 500 nm filling about 70-80% of the cell cytoplasm. Gas chromatography/mass spectrometry analyses of total cellular lipids and from isolated triacylglycerols (TAG) confirmed a similar fatty acid composition with a large portion of iso- and anteiso-methyl-branched fatty acids. De novo biosynthesis of wax esters (WE) appeared only during cocultivation on glucose and hexadecanol as carbon source. Homology alignments with the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; AtfA) from Acinetobacter baylyi strain ADP1 yielded one open reading frame in the genome databases of S. avermitilis MA-4680 referred to as SAV7256 with 25.3% homology. The highly conserved HHAxxDG active site motif found in AtfA, which is present in SAV7256, as well as the similar hydrophobicity profiles of AtfA and SAV7256 indicate a similar structure and function of both proteins. High acyl-CoA:diacylglycerol acyltransferase activity (DGAT; 143 pmol (mg min)(-1)) but low wax ester synthase activity (WS; 1.3 pmol (mg min)(-1)) were detected in crude extracts of S. avermitilis, which were consistent with the high TAG and negligible WE content of the cells. This indicates that TAG accumulation in S. avermitilis MA-4680 is mediated by the classical acyl-CoA-dependent DGAT pathway. Heterologous expression experiments in recombinant Escherichia coli BL21(DE3) demonstrated both WS and DGAT enzyme activity of SAV7256. Furthermore, substrate specificities of the acyltransferase SAV7256 will be discussed.

  20. Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family.

    Science.gov (United States)

    Uyama, Toru; Ikematsu, Natsuki; Inoue, Manami; Shinohara, Naoki; Jin, Xing-Hua; Tsuboi, Kazuhito; Tonai, Takeharu; Tokumura, Akira; Ueda, Natsuo

    2012-09-14

    Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.

  1. A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins.

    Science.gov (United States)

    Rinaldo, Amy R; Cavallini, Erika; Jia, Yong; Moss, Sarah M A; McDavid, Debra A J; Hooper, Lauren C; Robinson, Simon P; Tornielli, Giovanni B; Zenoni, Sara; Ford, Christopher M; Boss, Paul K; Walker, Amanda R

    2015-11-01

    Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.

  2. Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells

    DEFF Research Database (Denmark)

    Konitsiotis, Antonios D; Chang, Shu-Chun; Jovanović, Biljana

    2014-01-01

    Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC......) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh...

  3. Familial lecithin: cholesterol acyltransferase deficiency complicated with unconjugated hyperbilirubinemia and peripheral neuropathy. The first reported cases in the Far East.

    Science.gov (United States)

    Iwamoto, A; Naito, C; Teramoto, T; Kato, H; Kako, M; Kariya, T; Shimizu, T; Oka, H; Oda, T

    1978-01-01

    Three Japanese patients with lecithin: cholesterol acyltransferase (LCAT) deficiency, the offspring of a consanguineous marriage, are described. In addition to the characteristic clinical and laboratory findings of the disease, our patients had hitherto unreported manifestations, namely unconjugated hyperbilirubinemia, peripheral neuropathy and marked hypocholesterolemia. Although the mechanism of the unconjugated hyperbilirubinemia is not clear, the role of impaired hepatic bilirubin uridine-diphosphate-glucuronyl transferase activity combined with another unknown factor(s) was postulated. Non-random assortment was observed between LCAT deficiency and haptoglobin types, as previously reported. The discovery of Japanese patients with LCAT deficiency indicates that the distribution of this hereditary metabolic disorder is not confined to the Western hemisphere.

  4. Lecithin:Retinol Acyltransferase: A Key Enzyme Involved in the Retinoid (visual) Cycle.

    Science.gov (United States)

    Sears, Avery E; Palczewski, Krzysztof

    2016-06-07

    Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRAT mutations.

  5. Endocrine impact of Helicobacter pylori : Focus on ghrelin and ghrelin o -acyltransferase

    Institute of Scientific and Technical Information of China (English)

    Penny L Jeffery; Michael A McGuckin; Sara K Linden

    2011-01-01

    Ghrelin is predominantly produced by the gastric enteroendocrine cell compartment and is octanoylated by the recently discovered ghrelin o -acyltransferase (GOAT) before secretion into the bloodstream. This octanoylation is essential for many of the biological properties of ghrelin including appetite stimulation and anti-inflammatory properties as only the acylated form of ghrelin binds to the ghrelin receptor, the growth hormone secretagogue receptor (GHS-R). Given the gastric location of ghrelin production, it is perhaps not surprising that insult to the gastric mucosa affects circulating ghrelin levels in humans. Helicobacter pylori (H. pylori ) infects more than fifty percent of the world's population and once established within the gastric mucosa, can persist for life. Infection is associated with chronic gastritis, gastric atrophy and ulceration, reduced appetite and a lower body mass index (BMI). The large majority of studies investigating levels of circulating ghrelin and ghrelin expression in the stomach in patients with H. pylori infection indicate that the bacterium has a negative impact on ghrelin production and/or secretion. Eradication of infection restores ghrelin, improves appetite and increases BMI in some studies, however, a causative relationship between H. pylori -associated serum ghrelin decline and food intake and obesity has not been established. Most studies measure total ghrelin in the circulation although the measurement of the ratio of acyl/total ghrelin gives a clearer indication that the ghrelin acylation process is altered during infection and atrophy. GOAT is essential for the production of biologically-active, acyl ghrelin and the impact of H. pylori on GOAT expression and activity will be highly informative in the future.

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

    Science.gov (United States)

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

    1998-05-01

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

  7. Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus)

    Science.gov (United States)

    Blanco, Ayelén Melisa; Gómez-Boronat, Miguel; Alonso-Gómez, Ángel Luis; Yufa, Roman; Unniappan, Suraj; Delgado, María Jesús; Valenciano, Ana Isabel

    2017-01-01

    Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish. PMID:28178327

  8. Lysophosphatidic acid acyltransferase β (LPAATβ promotes the tumor growth of human osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Farbod Rastegar

    Full Text Available BACKGROUND: Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2 in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This

  9. The Mycobacterium tuberculosis Ag85A is a novel diacylglycerol acyltransferase involved in lipid body formation.

    Science.gov (United States)

    Elamin, Ayssar A; Stehr, Matthias; Spallek, Ralf; Rohde, Manfred; Singh, Mahavir

    2011-09-01

    Mycobacterium tuberculosis accumulates large amounts of triacylglycerol (TAG) which acts as storage compounds for energy and carbon. The mycobacterial triacylglycerols stored in the form of intracellular lipid droplets are essential for long-term survival of M. tuberculosis during a dormant state. We report here that when the M. tuberculosis mycolytransferase Ag85A is overexpressed in Mycobacterium smegmatis mc(2)155, cell morphology was changed and the cells became grossly enlarged. A massive formation of lipid bodies and a change in lipid pattern was observed simultaneously. We suspected a possible role of Ag85A in the acyl lipid metabolism and discovered that the enzyme possesses acyl-CoA:diacylglycerol acyltransferase (DGAT) activity in addition to its well-known function as mycolyltransferase. Ag85A mediates the transesterification of diacylglycerol using long-chain acyl-CoA as acyl donors. The K(m) and K(cat) values for palmitoleoyl-coenzyme A were 390 µM and 55.54 min(-1) respectively. A docking model suggests that palmitoleoyl-coenzyme A and 1,2-dipalmitin occupy the same active site as trehalose 6,6'-dimycolate and trehalose 6'-monomycolate. The site-directed Ser126Ala mutation of the active site proved that this residue is involved in the catalytic activity of this enzyme. Although not proven conclusively for dormant stage of M. tuberculosis, our novel finding about the synthesis of TAGs by Ag85A strongly suggests that Ag85A may play a significant role in the formation of lipid storage bodies and thus also in the establishment and maintenance of a persistent tuberculosis infection.

  10. Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues.

    Science.gov (United States)

    Cao, Heping; Shockey, Jay M; Klasson, K Thomas; Chapital, Dorselyn C; Mason, Catherine B; Scheffler, Brian E

    2013-01-01

    Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.

  11. Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas*

    Science.gov (United States)

    Boyle, Nanette R.; Page, Mark Dudley; Liu, Bensheng; Blaby, Ian K.; Casero, David; Kropat, Janette; Cokus, Shawn J.; Hong-Hermesdorf, Anne; Shaw, Johnathan; Karpowicz, Steven J.; Gallaher, Sean D.; Johnson, Shannon; Benning, Christoph; Pellegrini, Matteo; Grossman, Arthur; Merchant, Sabeeha S.

    2012-01-01

    Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions. PMID:22403401

  12. Yeast Gup1(2 Proteins Are Homologues of the Hedgehog Morphogens Acyltransferases HHAT(L: Facts and Implications

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    Cândida Lucas

    2016-11-01

    Full Text Available In multiple tissues, the Hedgehog secreted morphogen activates in the receiving cells a pathway involved in cell fate, proliferation and differentiation in the receiving cells. This pathway is particularly important during embryogenesis. The protein HHAT (Hedgehog O-acyltransferase modifies Hh morphogens prior to their secretion, while HHATL (Hh O-acyltransferase-like negatively regulates the pathway. HHAT and HHATL are homologous to Saccharomyces cerevisiae Gup2 and Gup1, respectively. In yeast, Gup1 is associated with a high number and diversity of biological functions, namely polarity establishment, secretory/endocytic pathway functionality, vacuole morphology and wall and membrane composition, structure and maintenance. Phenotypes underlying death, morphogenesis and differentiation are also included. Paracrine signalling, like the one promoted by the Hh pathway, has not been shown to occur in microbial communities, despite the fact that large aggregates of cells like biofilms or colonies behave as proto-tissues. Instead, these have been suggested to sense the population density through the secretion of quorum-sensing chemicals. This review focuses on Gup1/HHATL and Gup2/HHAT proteins. We review the functions and physiology associated with these proteins in yeasts and higher eukaryotes. We suggest standardisation of the presently chaotic Gup-related nomenclature, which includes KIAA117, c3orf3, RASP, Skinny, Sightless and Central Missing, in order to avoid the disclosure of otherwise unnoticed information.

  13. Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nepsilon-acyltransferase involved in mycobactin biosynthesis.

    Science.gov (United States)

    Frankel, Brenda A; Blanchard, John S

    2008-09-15

    Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine N(epsilon)-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of k(cat)/K(m) revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His(130) and Asp(168) indicated that both residues are critical for acyltransferase activity and suggests that His(130) is responsible for general base activation of the epsilon-amino group of lysine.

  14. Structural basis for selective recognition of acyl chains by the membrane-associated acyltransferase PatA.

    Science.gov (United States)

    Albesa-Jové, David; Svetlíková, Zuzana; Tersa, Montse; Sancho-Vaello, Enea; Carreras-González, Ana; Bonnet, Pascal; Arrasate, Pedro; Eguskiza, Ander; Angala, Shiva K; Cifuente, Javier O; Korduláková, Jana; Jackson, Mary; Mikušová, Katarína; Guerin, Marcelo E

    2016-03-11

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design.

  15. The unprocessed preprotein form IAT(C103S) of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing

    NARCIS (Netherlands)

    Garcia-Estrada, Carlos; Vaca, Inmaculada; Fierro, Francisco; Sjollema, Klaas; Veenhuis, Marten; Francisco Martin, Juan

    2008-01-01

    Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant

  16. Synthesis of Penicillium chrysogenum acetyl-CoA : Isopenicillin N acyltransferase in Hansenula polymorpha: First step towards the introduction of a new metabolic pathway

    NARCIS (Netherlands)

    Lutz, Marco V.; Bovenberg, Roel A.L.; Klei, Ida J. van der; Veenhuis, Marten

    2005-01-01

    The enzyme acetyl-CoA:isopenicillin N acyltransferase (IAT) is a peroxisomal enzyme that mediates the final step of penicillin biosynthesis in the filamentous fungi Penicillium chrysogenum and Aspergillus nidulans. However, the precise role of peroxisomes in penicillin biosynthesis is still not clea

  17. Higher high density lipoprotein cholesterol associated with moderate alcohol consumption is not related to altered plasma lecithin : cholesterol acyltransferase and lipid transfer protein activity levels

    NARCIS (Netherlands)

    Riemens, SC; vanTol, A; Hoogenberg, K; vanGent, T; Scheek, LM; Sluiter, WJ; Dullaart, RPF

    1997-01-01

    Lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are important factors involved in HDL metabolism. Altered plasma activity levels of these factors could play a role in the increase in high density lipoprotein (HDL) choles

  18. Effects of the diacylglycerol o-acyltransferase 1 (DGAT1) K232A polymorphism on fatty acid, protein, and mineral composition of dairy cattle milk

    NARCIS (Netherlands)

    Bovenhuis, H.; Visker, M.H.P.W.; Poulsen, N.A.; Sehested, J.; Valenberg, van H.J.F.; Arendonk, van J.A.M.; Larsen, L.B.; Buitenhuis, A.J.

    2016-01-01

    Several studies have described associations between the diacylglycerol o-acyltransferase 1 (DGAT1) K232A polymorphism and routinely collected milk production traits but not much is known about effects of the DGAT1 polymorphism on detailed milk composition. The aim of this study was to estimate ef

  19. Ghrelin O-acyltransferase (GOAT), a specific enzyme that modifies ghrelin with a medium-chain fatty acid.

    Science.gov (United States)

    Kojima, Masayasu; Hamamoto, Akie; Sato, Takahiro

    2016-10-01

    In the gastric peptide hormone ghrelin, serine 3 (threonine 3 in frogs) is modified, primarily by n-octanoic acid; this modification is essential for ghrelin's activity. The enzyme that transfers n-octanoic acid to Ser3 of ghrelin is ghrelin O-acyltransferase (GOAT). GOAT, the only enzyme known to catalyze acyl modification of ghrelin, specifically modifies serine (or threonine) at the third position and does not modify other serine residues in ghrelin peptides. GOAT prefers n-hexanoyl-CoA over n-octanoyl-CoA as the acyl donor, although in the stomach the n-octanoyl form is the predominant form of acyl-modified ghrelin. GOAT is a promising target for drug development to treat metabolic diseases and eating disorders.

  20. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics.

    Science.gov (United States)

    Tschapalda, Kirsten; Zhang, Ya-Qin; Liu, Li; Golovnina, Kseniya; Schlemper, Thomas; Eichmann, Thomas O; Lal-Nag, Madhu; Sreenivasan, Urmila; McLenithan, John; Ziegler, Slava; Sztalryd, Carole; Lass, Achim; Auld, Douglas; Oliver, Brian; Waldmann, Herbert; Li, Zhuyin; Shen, Min; Boxer, Matthew B; Beller, Mathias

    2016-06-01

    Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  1. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ.

    Science.gov (United States)

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A; Formiggini, Fabio; Polishchuk, Roman S; Corda, Daniela; Luini, Alberto

    2016-07-12

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself).

  2. Kinetic mechanism and order of substrate binding for sn-glycerol-3-phosphate acyltransferase from squash (Cucurbita moschata).

    Science.gov (United States)

    Hayman, Matthew W; Fawcett, Tony; Slabas, Antoni R

    2002-03-13

    sn-Glycerol-3-phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three-dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451-453; Turnbull et al. (2001) Structure 9, 347-353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl-acyl carrier protein (acyl-ACP) substrates. The reaction proceeds via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate. We have also determined that the reaction will proceed with C(4:0)-CoA, C(6:0)-CoA and C(12:0)-ACP substrates, allowing a wider choice of acyl groups for future co-crystallisation studies.

  3. Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity.

    Science.gov (United States)

    Hayman, M W; Fawcett, T; Schierer, T F; Simon, J W; Kroon, J T; Gilroy, J S; Rice, D W; Rafferty, J; Turnbull, A P; Sedelnikova, S E; Slabas, A R

    2000-12-01

    In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.

  4. Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata).

    Science.gov (United States)

    Turnbull, A P; Rafferty, J B; Sedelnikova, S E; Slabas, A R; Schierer, T P; Kroon, J T; Nishida, I; Murata, N; Simon, J W; Rice, D W

    2001-03-01

    Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system.

  5. A Class of Diacylglycerol Acyltransferase 1 Inhibitors Identified by a Combination of Phenotypic High-throughput Screening, Genomics, and Genetics

    Directory of Open Access Journals (Sweden)

    Kirsten Tschapalda

    2016-06-01

    Full Text Available Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened >320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1, a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests.

  6. Isolation and expression analysis of glycerol-3-phosphate acyltransferase genes from peanuts (Arachis hypogaea L.

    Directory of Open Access Journals (Sweden)

    Chi, X.

    2015-09-01

    Full Text Available sn-Glycerol-3-phosphate acyltransferase (GPAT catalyzes the committed step in the production of glycerolipids. The functions of GPAT genes have been intensively studied in Arabidopsis, but not in peanuts (Arachis hypogaea L.. In this study, six AhGPAT genes were isolated from peanuts. Quantitative real-time RT-PCR analysis indicated that the AhGPAT9 transcript was more abundant in the stems, flowers, and seeds, whereas the transcript abundances of five other genes were higher in the leaves or flowers than in the other tissues examined. During seed development, the transcript levels of AhGPAT9 gradually increased, whereas the transcript levels of the other five genes decreased. In addition, the levels of AhGPAT2 transcript were distinctly enhanced after exposure to all four kinds of stress treatments except for ABA-treated leaves. The transcripts of AhGPAT1, AhGPAT6, AhGPAT8 and AhATS1 increased substantially in roots exposed to salt, drought, and ABA stress. The expressions of AhGPAT6, AhGPAT8, AhGPAT9 and AhATS1 were slightly higher in leaves under certain stress conditions than under normal conditions. The present study provides significant information for modifying oil deposition and improving the abiotic stress resistance of peanuts through molecular breeding.La aciltransferasa sn-glicerol-3-fosfato (ATGP cataliza el comprometido paso de la producción de glicerolípidos. Las funciones de los genes AhATGP se han estudiado intensivamente en Arabidopsis, pero no en cacahuete (Arachis hypogaea L.. En este estudio, seis genes AhATGP se aislaron a partir de cacahuetes. El análisis a tiempo real RT-PCR cuantitativa indicó que la transcripción AhATGP9 fue más abundante en tallos, flores y semillas, mientras que la abundancia de la transcripción de los otros cinco genes fueron mayores en hojas o flores que en los otros tejidos examinados. Durante el desarrollo de la semilla, los niveles de transcripción de AhATGP9 aumentaron gradualmente

  7. Phylogenetic analysis of glycerol 3-phosphate acyltransferases in opisthokonts reveals unexpected ancestral complexity and novel modern biosynthetic components.

    Directory of Open Access Journals (Sweden)

    Heather C Smart

    Full Text Available Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT, have been described, and characterized primarily in model organisms like Saccharomyces cerevisiae and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of 'fungal' orthologs in the basal taxa of the holozoa and 'animal' orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens.

  8. The Arabidopsis DCR encoding a soluble BAHD acyltransferase is required for cutin polyester formation and seed hydration properties.

    Science.gov (United States)

    Panikashvili, David; Shi, Jian Xin; Schreiber, Lukas; Aharoni, Asaph

    2009-12-01

    The cuticle covering every plant aerial organ is largely made of cutin that consists of fatty acids, glycerol, and aromatic monomers. Despite the huge importance of the cuticle to plant development and fitness, our knowledge regarding the assembly of the cutin polymer and its integration in the complete cuticle structure is limited. Cutin composition implies the action of acyltransferase-type enzymes that mediate polymer construction through ester bond formation. Here, we show that a member of the BAHD family of acyltransferases (DEFECTIVE IN CUTICULAR RIDGES [DCR]) is required for incorporation of the most abundant monomer into the polymeric structure of the Arabidopsis (Arabidopsis thaliana) flower cutin. DCR-deficient plants display phenotypes that are typically associated with a defective cuticle, including altered epidermal cell differentiation and postgenital organ fusion. Moreover, levels of the major cutin monomer in flowers, 9(10),16-dihydroxy-hexadecanoic acid, decreased to an almost undetectable amount in the mutants. Interestingly, dcr mutants exhibit changes in the decoration of petal conical cells and mucilage extrusion in the seed coat, both phenotypes formerly not associated with cutin polymer assembly. Excessive root branching displayed by dcr mutants and the DCR expression pattern in roots pointed to the function of DCR belowground, in shaping root architecture by influencing lateral root emergence and growth. In addition, the dcr mutants were more susceptible to salinity, osmotic, and water deprivation stress conditions. Finally, the analysis of DCR protein localization suggested that cutin polymerization, possibly the oligomerization step, is partially carried out in the cytoplasmic space. Therefore, this study extends our knowledge regarding the functionality of the cuticular layer and the formation of its major constituent the polymer cutin.

  9. Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

    Science.gov (United States)

    Villa, Juan A.; Cabezas, Matilde; de la Cruz, Fernando

    2014-01-01

    Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids. PMID:24296496

  10. Synthesis of Penicillium chrysogenum acetyl-CoA: Isopenicillin N acyltransferase in Hansenula polymorpha: First step towards the introduction of a new metabolic pathway

    OpenAIRE

    Lutz, Marco V.; Bovenberg, Roel A. L.; van der Klei, Ida J.; Veenhuis, Marten

    2005-01-01

    The enzyme acetyl-CoA:isopenicillin N acyltransferase (IAT) is a peroxisomal enzyme that mediates the final step of penicillin biosynthesis in the filamentous fungi Penicillium chrysogenum and Aspergillus nidulans. However, the precise role of peroxisomes in penicillin biosynthesis is still not clear. To be able to use the power of yeast genetics to solve the function of peroxisomes in penicillin biosynthesis, we introduced IAT in the yeast Hansenula polymorpha. To this purpose, the P. chryso...

  11. Iridoid enriched fraction from Ajuga iva reduce cholesterolemia, triacylglycerolemia and increase the lecithin:cholesterol acyltransferase activity of rats fed a cholesterol-rich diet

    OpenAIRE

    Marie A. Lacaille-Dubois; Josiane Prost; Sherazede Bouderbala; Malika Bouchenak

    2012-01-01

    Objective: In this study, we examined the effect of iridoid (I) derived from lyophilized aqueous extract of Ajuga iva on serum HDL2 and HDL3 compositions and lecithin:cholesterol acyltransferase (LCAT) activity, enzyme responsible for reverse cholesterol transport. Methods: Male Wistar rats (n=24) weighing 120±5 g were fed a diet containing 1% cholesterol-rich diet for 15 days. After this phase, the hypercholesterolemic (HC) rats were divided into groups fed the same diet and received or...

  12. Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters.

    Science.gov (United States)

    Mañas-Fernández, Aurora; Li-Beisson, Yonghua; Alonso, Diego López; García-Maroto, Federico

    2010-09-01

    The glycerol-based lipid polyester called cutin is a main component of cuticle, the protective interface of aerial plant organs also controlling compound exchange with the environment. Though recent progress towards understanding of cutin biosynthesis has been made in Arabidopsis thaliana, little is known in other plants. One key step in this process is the acyl transfer reaction to the glycerol backbone. Here we report the cloning and molecular characterization of EpGPAT1, a gene encoding a glycerol-3-phosphate O-acyltransferase (GPAT) from Echium pitardii (Boraginaceae) with high similarity to the AtGPAT4/AtGPAT8 of Arabidopsis. Quantitative analysis by qRT-PCR showed highest expression of EpGPAT1 in seeds, roots, young leaves and flowers. Acyltransferase activity of EpGPAT1 was evidenced by heterologous expression in yeast. Ectopic expression in leaves of tobacco plants lead to an increase of C16 and C18 hydroxyacids and alpha,omega-diacids in the cell wall fraction, indicating a role in the biosynthesis of polyesters. Analysis of the genomic organization in Echium revealed the presence of EpGPAT2, a closely related gene which was found to be mostly expressed in developing leaves and flowers. The presence of a conserved HAD-like domain at the N-terminal moiety of GPATs from Echium, Arabidopsis and other plant species suggests a possible phosphohydrolase activity in addition to the reported acyltransferase activity. Evolutive implications of this finding are discussed.

  13. Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

    Science.gov (United States)

    Ngo, Alice; Fong, Kai T; Cox, Daniel L; Chen, Xi; Fisher, Andrew J

    2015-05-01

    Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

  14. Biosynthesis of Rhizobium meliloti lipooligosaccharide Nod factors: NodA is required for an N-acyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Atkinson, E.M.; Long, S.R. (Stanford Univ., CA (United States)); Palcic, M.M.; Hindsgaul, O. (Univ. of Alberta, Edmonton (Canada))

    1994-08-30

    Rhizobium bacteria synthesize N-acylated [beta]-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare [sup 35]S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-[beta]1,4-(GlcNac)[sub 3], by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase. 31 refs., 6 figs.

  15. The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana.

    Science.gov (United States)

    Wang, Hongyang; Sun, Chunlian; Jiang, Rui; He, Qin; Yang, Yu; Tian, Zhejuan; Tian, Zhendong; Xie, Conghua

    2014-07-01

    Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production.

  16. Small Intestine but Not Liver Lysophosphatidylcholine Acyltransferase 3 (Lpcat3) Deficiency Has a Dominant Effect on Plasma Lipid Metabolism.

    Science.gov (United States)

    Kabir, Inamul; Li, Zhiqiang; Bui, Hai H; Kuo, Ming-Shang; Gao, Guangping; Jiang, Xian-Cheng

    2016-04-01

    Lysophosphatidylcholine acyltransferase 3 (Lpcat3) is involved in phosphatidylcholine remodeling in the small intestine and liver. We investigated lipid metabolism in inducible intestine-specific and liver-specificLpcat3gene knock-out mice. We producedLpcat3-Flox/villin-Cre-ER(T2)mice, which were treated with tamoxifen (at days 1, 3, 5, and 7), to deleteLpcat3specifically in the small intestine. At day 9 after the treatment, we found that Lpcat3 deficiency in enterocytes significantly reduced polyunsaturated phosphatidylcholines in the enterocyte plasma membrane and reduced Niemann-Pick C1-like 1 (NPC1L1), CD36, ATP-binding cassette transporter 1 (ABCA1), and ABCG8 levels on the membrane, thus significantly reducing lipid absorption, cholesterol secretion through apoB-dependent and apoB-independent pathways, and plasma triglyceride, cholesterol, and phospholipid levels, as well as body weight. Moreover, Lpcat3 deficiency does not cause significant lipid accumulation in the small intestine. We also utilized adenovirus-associated virus-Cre to depleteLpcat3in the liver. We found that liver deficiency only reduces plasma triglyceride levels but not other lipid levels. Furthermore, there is no significant lipid accumulation in the liver. Importantly, small intestine Lpcat3 deficiency has a much bigger effect on plasma lipid levels than that of liver deficiency. Thus, inhibition of small intestine Lpcat3 might constitute a novel approach for treating hyperlipidemia.

  17. Biodiesel production from crude jatropha oil catalyzed by immobilized lipase/acyltransferase from Candida parapsilosis in aqueous medium.

    Science.gov (United States)

    Rodrigues, Joana; Perrier, Véronique; Lecomte, Jérôme; Dubreucq, Eric; Ferreira-Dias, Suzana

    2016-10-01

    The lipase/acyltransferase from Candida parapsilosis (CpLIP2) immobilized on two synthetic resins (Accurel MP 1000 and Lewatit VP OC 1600) was used as catalyst for the production of biodiesel (fatty acid methyl esters, FAME) by transesterification of jatropha oil with methanol, in a lipid/aqueous system. The oil was dispersed in a buffer solution (pH 6.5) containing methanol in excess (2M in the biphasic system; molar ratio methanol/acyl chains 2:1). Transesterification was carried out at 30°C, under magnetic stirring, using 10% (w/w) of immobilized enzyme in relation to oil. The maximum FAME yields were attained after 8h reaction time: 80.5% and 93.8%, when CpLIP2 immobilized on Accurel MP 1000 or on Lewatit VP OC 1600 were used, respectively. CpLIP2 on both Accurel MP 1000 and Lewatit VP OC 1600 showed high operational stability along 5 consecutive 8h batches.

  18. Knockdown of lecithin retinol acyltransferase increases all-trans retinoic acid levels and restores retinoid sensitivity in malignant melanoma cells.

    Science.gov (United States)

    Amann, Philipp M; Czaja, Katharina; Bazhin, Alexandr V; Rühl, Ralph; Skazik, Claudia; Heise, Ruth; Marquardt, Yvonne; Eichmüller, Stefan B; Merk, Hans F; Baron, Jens M

    2014-11-01

    Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

  19. Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein.

    Science.gov (United States)

    Amengual, Jaume; Golczak, Marcin; Palczewski, Krzysztof; von Lintig, Johannes

    2012-07-13

    Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.

  20. SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase

    Directory of Open Access Journals (Sweden)

    Martin Pagac

    2016-11-01

    Full Text Available Berardinelli-Seip congenital lipodystrophy 2 (BSCL2 is caused by loss-of-function mutations in SEIPIN, a protein implicated in both adipogenesis and lipid droplet expansion but whose molecular function remains obscure. Here, we identify physical and functional interactions between SEIPIN and microsomal isoforms of glycerol-3-phosphate acyltransferase (GPAT in multiple organisms. Compared to controls, GPAT activity was elevated in SEIPIN-deficient cells and tissues and GPAT kinetic values were altered. Increased GPAT activity appears to underpin the block in adipogenesis and abnormal lipid droplet morphology associated with SEIPIN loss. Overexpression of Gpat3 blocked adipogenesis, and Gpat3 knockdown in SEIPIN-deficient preadipocytes partially restored differentiation. GPAT overexpression in yeast, preadipocytes, and fly salivary glands also formed supersized lipid droplets. Finally, pharmacological inhibition of GPAT in Seipin−/− mouse preadipocytes partially restored adipogenesis. These data identify SEIPIN as an evolutionarily conserved regulator of microsomal GPAT and suggest that GPAT inhibitors might be useful for the treatment of human BSCL2 patients.

  1. Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation.

    Science.gov (United States)

    Rebeil, Roberto; Ernst, Robert K; Jarrett, Clayton O; Adams, Kristin N; Miller, Samuel I; Hinnebusch, B Joseph

    2006-02-01

    Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37 degrees C, tetra-acylated lipid A is the major form; but at 26 degrees C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C12 and C(16:1) groups, respectively, to lipid IV(A) to generate the hexa-acylated form, and that their expression is upregulated at 21 degrees C in vitro and in the flea midgut. A Y. pestis deltamsbB deltalpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.

  2. Sterol O-Acyltransferase 2-Driven Cholesterol Esterification Opposes Liver X Receptor-Stimulated Fecal Neutral Sterol Loss.

    Science.gov (United States)

    Warrier, Manya; Zhang, Jun; Bura, Kanwardeep; Kelley, Kathryn; Wilson, Martha D; Rudel, Lawrence L; Brown, J Mark

    2016-02-01

    Statin drugs have proven a successful and relatively safe therapy for the treatment of atherosclerotic cardiovascular disease (CVD). However, even with the substantial low-density lipoprotein (LDL) cholesterol lowering achieved with statin treatment, CVD remains the top cause of death in developed countries. Selective inhibitors of the cholesterol esterifying enzyme sterol-O acyltransferase 2 (SOAT2) hold great promise as effective CVD therapeutics. In mouse models, previous work has demonstrated that either antisense oligonucleotide (ASO) or small molecule inhibitors of SOAT2 can effectively reduce CVD progression, and even promote regression of established CVD. Although it is well known that SOAT2-driven cholesterol esterification can alter both the packaging and retention of atherogenic apoB-containing lipoproteins, here we set out to determine whether SOAT2-driven cholesterol esterification can also impact basal and liver X receptor (LXR)-stimulated fecal neutral sterol loss. These studies demonstrate that SOAT2 is a negative regulator of LXR-stimulated fecal neutral sterol loss in mice.

  3. Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols.

    Science.gov (United States)

    Haïli, Nawel; Louap, Julien; Canonge, Michel; Jagic, Franjo; Louis-Mondésir, Christelle; Chardot, Thierry; Briozzo, Pierre

    2016-01-01

    The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications.

  4. Divergence in the enzymatic activities of a tomato and Solanum pennellii alcohol acyltransferase impacts fruit volatile ester composition.

    Science.gov (United States)

    Goulet, Charles; Kamiyoshihara, Yusuke; Lam, Nghi B; Richard, Théo; Taylor, Mark G; Tieman, Denise M; Klee, Harry J

    2015-01-01

    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species Solanum pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrate preferences that explain the variations observed in the volatiles. The results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of the synthesis and degradation of esters.

  5. Single Nucleotide Polymorphisms (SNPs in Exon 6 of Lecithin Cholesterol Acyltransferase (LCAT Gene in Indonesian Local Sheep

    Directory of Open Access Journals (Sweden)

    Hidayati

    2014-08-01

    Full Text Available Lecithin cholesterol acyltransferase (LCAT is a soluble enzyme that converts cholesterol and lecithin to cholesteryl esters and lysolecithins on the surface of high density lipoprotein and plays an important role in lipoprotein metabolism. The research was aimed to explore single nucleotide polymorphisms of LCAT gene in Indonesian local sheep. A total of 118 genomic DNA of Indonesian local sheep were used in this research, consisted of Sumatera Thin Tail (43 heads, Garut (19 heads, Javanese Thin Tail (17 heads, Javanese Fat Tail (6 heads, Rote Island (7 heads, Kissar (7 heads, Sumbawa (10 heads, and Lembah Palu (9 heads. Polymerase chain reaction was used to amplify genomic DNA for exon 6 (250 bp and direct sequencing method was used to identify polymorphism sequences. The sequences were analyzed with BioEdit and MEGA 5.2 software. The BLAST sequence was obtained from Gene Bank GQ 150556.1. The results showed three novel SNPs, i.e. c.742C>T, c.770 T>A and c.882C>T. Substitution of cytosine to thymine c.742 is a synonymous mutation; thymine to adenine c.770 and cytosine to thymine c.882 are non-synonymous mutations. Polymorphisms of LCAT gene exon 6 was found in Sumatera Thin Tail, Javanese Thin Tail, Javanese Fat Tail, Garut, Lembah Palu, and Rote Island.

  6. Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols.

    Directory of Open Access Journals (Sweden)

    Elizabeth R Cattaneo

    Full Text Available BACKGROUND: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT. In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. METHODS AND RESULTS: Incubation of GPAT2-transfected CHO-K1 cells with [1-(14C]arachidonate for 3 h increased incorporation of [(14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. CONCLUSIONS: These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

  7. Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments.

    Science.gov (United States)

    Lanfranconi, Mariana P; Alvarez, Adrián F; Alvarez, Héctor M

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest.

  8. Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: a moon lighting property.

    Science.gov (United States)

    Baghel, Anil S; Tandon, Rashmi; Gupta, Garima; Kumar, Ajit; Sharma, Raman K; Aggarwal, Neha; Kathuria, Abha; Saini, Neeraj K; Bose, Mridula; Prasad, Ashok K; Sharma, Sunil K; Nath, Mahendra; Parmar, Virinder S; Raj, Hanumantharao G

    2011-12-20

    The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.

  9. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase

    Directory of Open Access Journals (Sweden)

    Thomas Lanyon-Hogg

    2016-06-01

    Full Text Available In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI” class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a, RU-SKI 43 (9b, RU-SKI 101 (9c, and RU-SKI 201 (9d were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015 [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

  10. Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.).

    Science.gov (United States)

    Sun, Li; Ouyang, Chao; Kou, Shanglong; Wang, Shenghua; Yao, Yunyi; Peng, Tong; Xu, Ying; Tang, Lin; Chen, Fang

    2011-01-01

    A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).

  11. Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

    Science.gov (United States)

    Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

    2015-03-01

    The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.

  12. Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds.

    Science.gov (United States)

    Ruiz-López, Noemí; Garcés, Rafael; Harwood, John L; Martínez-Force, Enrique

    2010-01-01

    The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis-Menten K(m) for the glycerol 3-phosphate was 354 muM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.

  13. Structural and Affinity Determinants in the Interaction between Alcohol Acyltransferase from F. x ananassa and Several Alcohol Substrates: A Computational Study.

    Science.gov (United States)

    Navarro-Retamal, Carlos; Gaete-Eastman, Carlos; Herrera, Raúl; Caballero, Julio; Alzate-Morales, Jans H

    2016-01-01

    Aroma and flavor are important factors of fruit quality and consumer preference. The specific pattern of aroma is generated during ripening by the accumulation of volatiles compounds, which are mainly esters. Alcohol acyltransferase (AAT) (EC 2.3.1.84) catalyzes the esterification reaction of aliphatic and aromatic alcohols and acyl-CoA into esters in fruits and flowers. In Fragaria x ananassa, there are different volatiles compounds that are obtained from different alcohol precursors, where octanol and hexanol are the most abundant during fruit ripening. At present, there is not structural evidence about the mechanism used by the AAT to synthesize esters. Experimental data attribute the kinetic role of this enzyme to 2 amino acidic residues in a highly conserved motif (HXXXD) that is located in the middle of the protein. With the aim to understand the molecular and energetic aspects of volatiles compound production from F. x ananassa, we first studied the binding modes of a series of alcohols, and also different acyl-CoA substrates, in a molecular model of alcohol acyltransferase from Fragaria x ananassa (SAAT) using molecular docking. Afterwards, the dynamical behavior of both substrates, docked within the SAAT binding site, was studied using routine molecular dynamics (MD) simulations. In addition, in order to correlate the experimental and theoretical data obtained in our laboratories, binding free energy calculations were performed; which previous results suggested that octanol, followed by hexanol, presented the best affinity for SAAT. Finally, and concerning the SAAT molecular reaction mechanism, it is suggested from molecular dynamics simulations that the reaction mechanism may proceed through the formation of a ternary complex, in where the Histidine residue at the HXXXD motif deprotonates the alcohol substrates. Then, a nucleophilic attack occurs from alcohol charged oxygen atom to the carbon atom at carbonyl group of the acyl CoA. This mechanism is in

  14. The Role of Lecithin: Retinol Acyltransferase (LRAT)-Mediated Esterification of Vitamin A in Regulating Human Breast Cancer Cell Proliferation and Differentiation

    Science.gov (United States)

    2007-04-01

    Rhim, J.S., Nanus, D.M., and Gudas, L.J. 2002. Retinol metabolism and lecithin:retinol acyltransferase levels are reduced in cultured human prostate...tested LRAT mRNA levels in Wt and RARγ -/- F9 cells, an epithelialtype of cell, which can synthesize retinyl esters. The RT-PCR analysis indicated...that LRAT expression was lost in the MDA-MB-231 breast cancer cells. Surprisingly, we did not observe RA associated increase in LRAT mRNA levels in

  15. Glycerol-3-phosphate acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor

    DEFF Research Database (Denmark)

    Zhang, Chongben; Cooper, Daniel E; Grevengoed, Trisha J

    2014-01-01

    Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might...... GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest...

  16. Mutations in Hedgehog acyltransferase (Hhat perturb Hedgehog signaling, resulting in severe acrania-holoprosencephaly-agnathia craniofacial defects.

    Directory of Open Access Journals (Sweden)

    Jennifer F Dennis

    Full Text Available Holoprosencephaly (HPE is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17% cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.

  17. Cloning, Characterization and Functional Analysis of Two Type 1 Diacylglycerol Acyltransferases (DGAT1s) from Tetraena mongolica

    Institute of Scientific and Technical Information of China (English)

    Minchun Li; Mingming Zhao; Hanying Wu; Wang Wu; Yinong Xu

    2013-01-01

    Two cDNAs encoding putative type 1 acyl-CoA:diacylglycerol acyltransferases (DGAT1,EC 2.3.1.20),were cloned from Tetraena mongolica Maxim.,an extreme xerophyte with high oil content in the stems.The 1,488-bp and 1,485-bp of the open reading frame (ORF) of the two cDNAs,designated as TmDGAT1a and TmDGAT1b,were both predicted to encode proteins of 495 and 494 amino acids,respectively.Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in the T.mongolica genome.In addition to ubiquitous expression with different intensity in different tissues,including stems,leaves and roots,TmDGAT1a and TmDGAT1b,were found to be strongly induced by high salinity,drought and osmotic stress,resulting in a remarkable increase of triacylglycerol (TAG) accumulation in T.mongolica plantlets.TmDGAT1a and TmDGAT1b activities were confirmed in the yeast H1246 quadruple mutant (DGA1,LRO1,ARE1,ARE2) by restoring DGAT activity of the mutant host to produce TAG.Overexpression.of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as in T.mongolica calli both resulted in an increase in oil content (ranging from 37% to 108%),accompanied by altered fatty acid profiles.

  18. The D519G Polymorphism of Glyceronephosphate O-Acyltransferase Is a Risk Factor for Familial Porphyria Cutanea Tarda

    Science.gov (United States)

    Farrell, Colin P.; Overbey, Jessica R.; Naik, Hetanshi; Nance, Danielle; McLaren, Gordon D.; McLaren, Christine E.; Zhou, Luming; Desnick, Robert J.; Parker, Charles J.

    2016-01-01

    Both familial and sporadic porphyria cutanea tarda (PCT) are iron dependent diseases. Symptoms of PCT resolve when iron stores are depleted by phlebotomy, and a sequence variant of HFE (C282Y, c.843G>A, rs1800562) that enhances iron aborption by reducing hepcidin expression is a risk factor for PCT. Recently, a polymorphic variant (D519G, c.1556A>G, rs11558492) of glyceronephosphate O-acyltransferase (GNPAT) was shown to be enriched in male patients with type I hereditary hemochromatosis (HFE C282Y homozygotes) who presented with a high iron phenotype, suggesting that GNPAT D519G, like HFE C282Y, is a modifier of iron homeostasis that favors iron absorption. To challenge this hypothesis, we investigated the frequency of GNPAT D519G in patients with both familial and sporadic PCT. Patients were screened for GNPAT D519G and allelic variants of HFE (both C282Y and H63D). Nucleotide sequencing of uroporphyrinogen decarboxylase (URO-D) identified mutant alleles. Patients with low erythrocyte URO-D activity or a damaging URO-D variant were classified as familial PCT (fPCT) and those with wild-type URO-D were classified as sporadic PCT (sPCT). GNPAT D519G was significantly enriched in the fPCT patient population (p = 0.0014) but not in the sPCT population (p = 0.4477). Both HFE C282Y and H63D (c.187C>G, rs1799945) were enriched in both PCT patient populations (p<0.0001) but showed no greater association with fPCT than with sPCT. Conclusion: GNPAT D519G is a risk factor for fPCT, but not for sPCT. PMID:27661980

  19. Vitamin A metabolism in benign and malignant melanocytic skin cells: importance of lecithin/retinol acyltransferase and RPE65.

    Science.gov (United States)

    Amann, Philipp M; Luo, Chonglin; Owen, Robert W; Hofmann, Claudia; Freudenberger, Muriel; Schadendorf, Dirk; Eichmüller, Stefan B; Bazhin, Alexandr V

    2012-02-01

    Disturbance in vitamin A metabolism seems to be an important attribute of cancer cells. Retinoids, particularly retinoic acid, have critical regulatory functions and appear to modulate tumor development and progression. The key step of vitamin A metabolism is the esterification of all-trans retinol, catalyzed by lecithin/retinol acyltransferase (LRAT). In this work, we show that malignant melanoma cells are able to esterify all-trans retinol and subsequently isomerize all-trans retinyl esters (RE) into 11-cis retinol, whereas their benign counterparts-melanocytes are not able to catalyze these reactions. Besides, melanoma cell lines express lecithin/retinol acyltranseferase both at the mRNA and protein levels. In contrast, melanocytes do not express this enzyme at the protein level, but mRNA of lecithin/retinol acyltransefrase could still be present at mRNA level. RPE65 is expressed in both melanocytic counterparts, and could be involved in the subsequent isomerization of RE produced by lecithin/retinol acyltransefrase to 11-cis retinol. Cellular retinol-binding protein 2 does not appear to be involved in the regulation of all-trans retinol esterification in these cells. Expression of LRAT and RPE65 can be modulated by retinoids. We propose that the post-transcriptional regulation of lecithin/retinol acyltransefrase could be involved in the differential expression of this enzyme. Besides, activities of LRAT and RPE65 may be important for removal of all-trans retinal which is the substrate for retinoic acid production in skin cells. Consequently, the decreasing cellular amount of retinoic acid and its precursor molecules could result in a change of gene regulation.

  20. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.

    Science.gov (United States)

    Dempsey, Daniel R; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J

    2015-11-01

    Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.

  1. Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds.

    Science.gov (United States)

    Kim, Hae Jin; Silva, Jillian E; Iskandarov, Umidjon; Andersson, Mariette; Cahoon, Rebecca E; Mockaitis, Keithanne; Cahoon, Edgar B

    2015-12-01

    Lysophosphatidic acid acyltransferase (LPAT) catalyzes acylation of the sn-2 position on lysophosphatidic acid by an acyl CoA substrate to produce the phosphatidic acid precursor of polar glycerolipids and triacylglycerols (TAGs). In the case of TAGs, this reaction is typically catalyzed by an LPAT2 from microsomal LPAT class A that has high specificity for C18 fatty acids containing Δ9 unsaturation. Because of this specificity, the occurrence of saturated fatty acids in the TAG sn-2 position is infrequent in seed oils. To identify LPATs with variant substrate specificities, deep transcriptomic mining was performed on seeds of two Cuphea species producing TAGs that are highly enriched in saturated C8 and C10 fatty acids. From these analyses, cDNAs for seven previously unreported LPATs were identified, including cDNAs from Cuphea viscosissima (CvLPAT2) and Cuphea avigera var. pulcherrima (CpuLPAT2a) encoding microsomal, seed-specific class A LPAT2s and a cDNA from C. avigera var. pulcherrima (CpuLPATB) encoding a microsomal, seed-specific LPAT from the bacterial-type class B. The activities of these enzymes were characterized in Camelina sativa by seed-specific co-expression with cDNAs for various Cuphea FatB acyl-acyl carrier protein thioesterases (FatB) that produce a variety of saturated medium-chain fatty acids. CvLPAT2 and CpuLPAT2a expression resulted in accumulation of 10:0 fatty acids in the Camelina sativa TAG sn-2 position, indicating a 10:0 CoA specificity that has not been previously described for plant LPATs. CpuLPATB expression generated TAGs with 14:0 at the sn-2 position, but not 10:0. Identification of these LPATs provides tools for understanding the structural basis of LPAT substrate specificity and for generating altered oil functionalities.

  2. Membrane topology of human monoacylglycerol acyltransferase-2 and identification of regions important for its localization to the endoplasmic reticulum.

    Science.gov (United States)

    McFie, Pamela J; Izzard, Sabrina; Vu, Huyen; Jin, Youzhi; Beauchamp, Erwan; Berthiaume, Luc G; Stone, Scot J

    2016-09-01

    Acyl CoA:2-monoacylglycerol acyltransferase (MGAT)-2 has an important role in dietary fat absorption in the intestine. MGAT2 resides in the endoplasmic reticulum and catalyzes the synthesis of diacylglycerol which is then utilized as a substrate for triacylglycerol synthesis. This triacylglycerol is then incorporated into chylomicrons which are released into the circulation. In this study, we determined the membrane topology of human MGAT2. Protease protection experiments showed that the C-terminus is exposed to the cytosol, while the N-terminus is partially buried in the ER membrane. MGAT2, like murine DGAT2, was found to have two transmembrane domains. We also identified a region of MGAT2 associated with the ER membrane that contains the histidine-proline-histidine-glycine sequence present in all DGAT2 family members that is thought to comprise the active site. Proteolysis experiments demonstrated that digestion of total cellular membranes from cells expressing MGAT2 with trypsin abolished MGAT activity, indicating that domains that are important for catalysis face the cytosol. We also explored the role that the five cysteines residues present in MGAT2 have in catalysis. MGAT activity was sensitive to two thiol modifiers, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). Furthermore, mutation of four cysteines resulted in a reduction in MGAT activity. However, when the C-terminal cysteine (C334) was mutated, MGAT activity was actually higher than that of wild-type FL-MGAT2. Lastly, we determined that both transmembrane domains of MGAT2 are important for its ER localization, and that MGAT2 is present in mitochondrial-associated membranes.

  3. Transcriptional and biochemical responses of monoacylglycerol acyltransferase-mediated oil synthesis and associated senescence-like responses in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Uday Kumar Divi

    2014-05-01

    Full Text Available Triacylglycerol (TAG accumulates in plant seeds as a major renewable source of carbon for food, fuel and industrial feedstock. Approaches to enhance TAG content by altering lipid pathways and genes in vegetative parts have gained significant attention for biofuel and other applications. However, consequences of these modifications are not always studied in detail. In an attempt to increase TAG levels in leaves we previously demonstrated that a novel substrate, monoacylglycerol (MAG, can be used for the biosynthesis of diacylglycerol (DAG and TAG. Transient expression of the Mus musculus monoacylglycerol acyltransferase MGAT1 and 2 in the model plant Nicotiana benthamiana increased TAG levels at 5 days post infiltration (dpi. Here we show that increased TAG and DAG levels can be achieved as early as 2 dpi. In addition, the MGAT1 infiltrated areas showed senescence-like symptoms from 3 dpi onwards. To unravel underlying molecular mechanisms, Illumina deep sequencing was carried out (a for de-novo assembling and annotation of N. benthamiana leaf transcripts and (b to characterize MGAT1-responsive transcriptome. We found that MGAT1-responsive genes are involved in several processes including TAG biosynthesis, photosynthesis, cell-wall, cutin, suberin, wax and mucilage biosynthesis, lipid and hormone metabolism. Comparative analysis with transcript profiles from other senescence studies identified characteristic gene expression changes involved in senescence induction. We confirmed that increased TAG and observed senescence-symptoms are due to the MAG depletion caused by MGAT1 activity and suggest a mechanism for MGAT1 induced TAG increase and senescence-like symptoms. The data generated will serve as a valuable resource for oil and senescence related studies and for future N. benthamiana transcriptome studies.

  4. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis.

    Science.gov (United States)

    Petit, Johann; Bres, Cécile; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Martin, Laetitia B B; Fich, Eric A; Joubès, Jérôme; Rose, Jocelyn K C; Domergue, Frédéric; Rothan, Christophe

    2016-06-01

    The thick cuticle covering and embedding the epidermal cells of tomato (Solanum lycopersicum) fruit acts not only as a protective barrier against pathogens and water loss but also influences quality traits such as brightness and postharvest shelf-life. In a recent study, we screened a mutant collection of the miniature tomato cultivar Micro-Tom and isolated several glossy fruit mutants in which the abundance of cutin, the polyester component of the cuticle, was strongly reduced. We employed a newly developed mapping-by-sequencing strategy to identify the causal mutation underlying the cutin deficiency in a mutant thereafter named gpat6-a (for glycerol-3-phosphate acyltransferase6). To this end, a backcross population (BC1F2) segregating for the glossy trait was phenotyped. Individuals displaying either a wild-type or a glossy fruit trait were then pooled into bulked populations and submitted to whole-genome sequencing prior to mutation frequency analysis. This revealed that the causal point mutation in the gpat6-a mutant introduces a charged amino acid adjacent to the active site of a GPAT6 enzyme. We further showed that this mutation completely abolished the GPAT activity of the recombinant protein. The gpat6-a mutant showed perturbed pollen formation but, unlike a gpat6 mutant of Arabidopsis (Arabidopsis thaliana), was not male sterile. The most striking phenotype was observed in the mutant fruit, where cuticle thickness, composition, and properties were altered. RNA sequencing analysis highlighted the main processes and pathways that were affected by the mutation at the transcriptional level, which included those associated with lipid, secondary metabolite, and cell wall biosynthesis.

  5. Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes.

    Science.gov (United States)

    Cooper, Daniel E; Grevengoed, Trisha J; Klett, Eric L; Coleman, Rosalind A

    2015-06-12

    Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4(-/-) mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4(-/-) mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4(-/-) mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4(-/-) BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4(-/-) brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.

  6. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

    Directory of Open Access Journals (Sweden)

    Cliona M Stapleton

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  7. Improvement of Neutral Lipid and Polyunsaturated Fatty Acid Biosynthesis by Overexpressing a Type 2 Diacylglycerol Acyltransferase in Marine Diatom Phaeodactylum tricornutum

    Directory of Open Access Journals (Sweden)

    Ying-Fang Niu

    2013-11-01

    Full Text Available Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT is a key enzyme that catalyzes the last step of triacylglyceride (TAG biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

  8. Key enzymes for biosynthesis of neutral lipid storage compounds in prokaryotes: properties, function and occurrence of wax ester synthases/acyl-CoA: diacylglycerol acyltransferases.

    Science.gov (United States)

    Wältermann, Marc; Stöveken, Tim; Steinbüchel, Alexander

    2007-02-01

    Triacylglycerols (TAGs) and wax esters (WEs) are beside polyhydroxyalkanoates (PHAs) important storage lipids in some groups of prokaryotes. Accumulation of these lipids occurs in cells when they are cultivated under conditions of unbalanced growth in the presence of high concentrations of a suitable carbon source, which can be used for fatty acid and storage lipid biosyntheses. The key enzymes, which mediate both WE and TAG formations from long-chain acyl-coenzyme A (CoA) as acyl donor and long-chain fatty alcohols or diacylglycerols as respective acyl acceptors in bacteria, are WE synthases/acyl-CoA:diacylglycerol acyltransferases (WS/DGATs). The WS/DGATs identified so far represent rather unspecific enzymes with broad spectra of possible substrates; this makes them interesting for many biotechnological applications. This review traces the molecular structure and biochemical properties including the probable regions responsible for acyltransferase properties, enzymatic activity and substrate specifities. The phylogenetic relationships based on amino acid sequence similarities of this unique class of enzymes were revealed. Furthermore, recent advances in understanding the physiological functions of WS/DGATs in their natural hosts including pathogenic Mycobacterium tuberculosis were discussed.

  9. Acyl-coenzyme A:cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7α-hydroxylase in cultured rat hepatocytes and in vivo in the rat

    NARCIS (Netherlands)

    Post, S.M.; Paul Zoeteweij, J.; Bos, M.H.A.; Wit, E.C.M. de; Havinga, R.; Kuipers, F.; Princen, H.M.G.

    1999-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors are currently in clinical development as potential lipid-lowering and antiatherosclerotic agents. We investigated the effect of avasimibe (C1- 1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7α- hydroxylase in cultur

  10. Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

    Science.gov (United States)

    Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

    1995-01-01

    Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

  11. Acyl-CoA:cholesterol acyltransferases (ACATs/SOATs): Enzymes with multiple sterols as substrates and as activators.

    Science.gov (United States)

    Rogers, Maximillian A; Liu, Jay; Song, Bao-Liang; Li, Bo-Liang; Chang, Catherine C Y; Chang, Ta-Yuan

    2015-07-01

    Cholesterol is essential to the growth and viability of cells. The metabolites of cholesterol include: steroids, oxysterols, and bile acids, all of which play important physiological functions. Cholesterol and its metabolites have been implicated in the pathogenesis of multiple human diseases, including: atherosclerosis, cancer, neurodegenerative diseases, and diabetes. Thus, understanding how cells maintain the homeostasis of cholesterol and its metabolites is an important area of study. Acyl-coenzyme A:cholesterol acyltransferases (ACATs, also abbreviated as SOATs) converts cholesterol to cholesteryl esters and play key roles in the regulation of cellular cholesterol homeostasis. ACATs are most unusual enzymes because (i) they metabolize diverse substrates including both sterols and certain steroids; (ii) they contain two different binding sites for steroidal molecules. In mammals, there are two ACAT genes that encode two different enzymes, ACAT1 and ACAT2. Both are allosteric enzymes that can be activated by a variety of sterols. In addition to cholesterol, other sterols that possess the 3-beta OH at C-3, including PREG, oxysterols (such as 24(S)-hydroxycholesterol and 27-hydroxycholesterol, etc.), and various plant sterols, could all be ACAT substrates. All sterols that possess the iso-octyl side chain including cholesterol, oxysterols, various plant sterols could all be activators of ACAT. PREG can only be an ACAT substrate because it lacks the iso-octyl side chain required to be an ACAT activator. The unnatural cholesterol analogs epi-cholesterol (with 3-alpha OH in steroid ring B) and ent-cholesterol (the mirror image of cholesterol) contain the iso-octyl side chain but do not have the 3-beta OH at C-3. Thus, they can only serve as activators and cannot serve as substrates. Thus, within the ACAT holoenzyme, there are site(s) that bind sterol as substrate and site(s) that bind sterol as activator; these sites are distinct from each other. These features form

  12. Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan.

    Science.gov (United States)

    Driessen, Nicole N; Stoop, Esther J M; Ummels, Roy; Gurcha, Sudagur S; Mishra, Arun K; Larrouy-Maumus, Gérald; Nigou, Jérôme; Gilleron, Martine; Puzo, Germain; Maaskant, Janneke J; Sparrius, Marion; Besra, Gurdyal S; Bitter, Wilbert; Vandenbroucke-Grauls, Christina M J E; Appelmelk, Ben J

    2010-11-01

    Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-(14)C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose

  13. Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design.

    Science.gov (United States)

    Jan, Anne-Hélène; Subileau, Maeva; Deyrieux, Charlotte; Perrier, Véronique; Dubreucq, Éric

    2016-02-01

    Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.

  14. The role of lecithin cholesterol acyltransferase and organic substances from coal in the etiology of Balkan endemic nephropathy: A new hypothesis

    Energy Technology Data Exchange (ETDEWEB)

    Pavlovic, N.M.; Orem, W.H.; Tatu, C.A.; Lerch, H.E.; Bunnell, J.E.; Feder, G.L.; Kostic, E.N.; Ordodi, V.L. [University of Nis, Nis (Serbia)

    2008-03-15

    Balkan endemic nephropathy (BEN) occurs in Serbia, Bulgaria, Romania, Bosnia and Herzegovina, and Croatia. BEN has been characterized as a chronic, slowly progressive renal disease of unknown etiology. In this study, we examined the influence of soluble organic compounds in drinking water leached from Pliocene lignite from BEN-endemic areas on plasma lecithin-cholesterol acyltransferase (LCAT) activity. We found that changes for all samples were the most prominent for the dilution category containing 90% plasma and 10% of diluting media. Water samples from BEN villages from Serbia and Romania showed higher LCAT inhibiting activity (P = 0.02) and (p = 0.003), respectively, compared to deionised water and non-endemic water. A secondary LCAT deficiency could result from this inhibitory effect of the organic compounds found in endemic water supplies and provide an ethiopathogenic basis for the development of BEN in the susceptible population.

  15. The role of lecithin cholesterol acyltransferase and organic substances from coal in the etiology of Balkan endemic nephropathy: A new hypothesis

    Science.gov (United States)

    Pavlovic, N.M.; Orem, W.H.; Tatu, C.A.; Lerch, H.E.; Bunnell, J.E.; Feder, G.L.; Kostic, E.N.; Ordodi, V.L.

    2008-01-01

    Balkan endemic nephropathy (BEN) occurs in Serbia, Bulgaria, Romania, Bosnia and Herzegovina, and Croatia. BEN has been characterized as a chronic, slowly progressive renal disease of unknown etiology. In this study, we examined the influence of soluble organic compounds in drinking water leached from Pliocene lignite from BEN-endemic areas on plasma lecithin-cholesterol acyltransferase (LCAT) activity. We found that changes for all samples were the most prominent for the dilution category containing 90% plasma and 10% of diluting media. Water samples from BEN villages from Serbia and Romania showed higher LCAT inhibiting activity (p = 0.02) and (p = 0.003), respectively, compared to deionised water and non-endemic water. A secondary LCAT deficiency could result from this inhibitory effect of the organic compounds found in endemic water supplies and provide an ethiopathogenic basis for the development of BEN in the susceptible population. ?? 2007 Elsevier Ltd. All rights reserved.

  16. Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

    Directory of Open Access Journals (Sweden)

    Laure Aymé

    Full Text Available Diacylglycerol acyltransferases (DGAT are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0. A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1 is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

  17. The enzyme lecithin-cholesterol acyltransferase esterifies cerebrosterol and limits the toxic effect of this oxysterol on SH-SY5Y cells.

    Science.gov (United States)

    La Marca, Valeria; Spagnuolo, Maria Stefania; Cigliano, Luisa; Marasco, Daniela; Abrescia, Paolo

    2014-07-01

    Cholesterol is mostly removed from the CNS by its conversion to cerebrosterol (24(S)-hydroxycholesterol, 24(S)OH-C), which is transported to the circulation for bile formation in liver. A neurotoxic role of this oxysterol was previously demonstrated in cell culture. Here, we provide evidence that the enzyme lecithin-cholesterol acyltransferase, long known to esterify cholesterol, also produces monoesters of 24(S)OH-C. Proteoliposomes containing apolipoprotein A-I or apolipoprotein E were used to stimulate the enzyme activity and entrap the formed esters. Proteoliposomes with apolipoprotein A-I were found to be more active than those with apolipoprotein E in stimulating the production of oxysteryl esters. Cholesterol and 24(S)OH-C were found to compete for enzyme activity. High levels of haptoglobin, as those circulating during the acute inflammatory phase, inhibited 24(S)OH-C esterification. When highly neurotoxic 24(S)OH-C was treated with enzyme and proteoliposomes before incubation with differentiated SH-SY5Y cells, the neuron survival improved. The esters of 24(S)OH-C, embedded into proteoliposomes by the enzyme and isolated from unesterified 24(S)OH-C by gel filtration chromatography, did not enter the neurons in culture. These results suggest that the enzyme, in the presence of the apolipoproteins, converts 24(S)OH-C into esters restricted to the extracellular environment, thus preventing or limiting oxysterol-induced neurotoxic injuries to neurons in culture. 24-hydroxycholesterol (24(S)OH-C) is neurotoxic. The enzyme lecithin-cholesterol acyltransferase (LCAT) synthesizes monoesters of 24(S)OH-C in reaction mixtures with proteoliposomes containing phospholipids and apolipoprotein A-I or apolipoprotein E. The esters, also produced by incubation of cerebrospinal fluid only with tritiated 24(S)OH-C, are embedded into lipoproteins that do not enter neurons in culture. The enzyme activity limits the toxicity of 24-hydroxycholesterol in neuron culture.

  18. Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica.

    Science.gov (United States)

    Aymé, Laure; Jolivet, Pascale; Nicaud, Jean-Marc; Chardot, Thierry

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.

  19. 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.

    Science.gov (United States)

    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.

  20. 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

    Energy Technology Data Exchange (ETDEWEB)

    Xing, Xiaomang; Li, Danyang; Chen, Dilong; Zhou, Liang [Faculty of Basic Medical Sciences, Chongqing Medical University, Chongqing 400016 China (China); Chonan, Ritsu [Koei Kogyo Co., Ltd., Tokyo, 101-0063 Japan (Japan); Yamahara, Johji [Pharmafood Institute, Kyoto, 602-8136 Japan (Japan); Wang, Jianwei, E-mail: wangjianwei1968@gmail.com [Department of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016 China (China); Li, Yuhao, E-mail: yuhao@sitcm.edu.au [Endocrinology and Metabolism Group, Sydney Institute of Health Sciences/Sydney Institute of Traditional Chinese Medicine, NSW 2000 Australia (Australia)

    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

  1. Intestine-specific deletion of acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2 protects mice from diet-induced obesity and glucose intolerance.

    Science.gov (United States)

    Nelson, David W; Gao, Yu; Yen, Mei-I; Yen, Chi-Liang Eric

    2014-06-20

    The absorption of dietary fat involves the re-esterification of digested triacylglycerol in the enterocytes, a process catalyzed by acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2. Mice without a functional gene encoding MGAT2 (Mogat2(-/-)) are protected from diet-induced obesity. Surprisingly, these mice absorb normal amounts of dietary fat but increase their energy expenditure. MGAT2 is expressed in tissues besides intestine, including adipose tissue in both mice and humans. To test the hypothesis that intestinal MGAT2 regulates systemic energy balance, we generated and characterized mice deficient in MGAT2 specifically in the small intestine (Mogat2(IKO)). We found that, like Mogat2(-/-) mice, Mogat2(IKO) mice also showed a delay in fat absorption, a decrease in food intake, and a propensity to use fatty acids as fuel when first exposed to a high fat diet. Mogat2(IKO) mice increased energy expenditure although to a lesser degree than Mogat2(-/-) mice and were protected against diet-induced weight gain and associated comorbidities, including hepatic steatosis, hypercholesterolemia, and glucose intolerance. These findings illustrate that intestinal lipid metabolism plays a crucial role in the regulation of systemic energy balance and may be a feasible intervention target. In addition, they suggest that MGAT activity in extraintestinal tissues may also modulate energy metabolism.

  2. Synthesis of Penicillium chrysogenum acetyl-CoA:isopenicillin N acyltransferase in Hansenula polymorpha: first step towards the introduction of a new metabolic pathway.

    Science.gov (United States)

    Lutz, Marco V; Bovenberg, Roel A L; van der Klei, Ida J; Veenhuis, Marten

    2005-11-01

    The enzyme acetyl-CoA:isopenicillin N acyltransferase (IAT) is a peroxisomal enzyme that mediates the final step of penicillin biosynthesis in the filamentous fungi Penicillium chrysogenum and Aspergillus nidulans. However, the precise role of peroxisomes in penicillin biosynthesis is still not clear. To be able to use the power of yeast genetics to solve the function of peroxisomes in penicillin biosynthesis, we introduced IAT in the yeast Hansenula polymorpha. To this purpose, the P. chrysogenum penDE gene, encoding IAT, was amplified from a cDNA library to eliminate the three introns and introduced in H. polymorpha. In this organism IAT protein was produced as a 40 kDa pre-protein and, as in P. chrysogenum, processed into an 11 and 29 kDa subunit, although the efficiency of processing seemed to be slightly reduced relative to P. chrysogenum. The P. chrysogenum IAT, produced in H. polymorpha, is normally localized in peroxisomes and in cell-free extracts IAT activity could be detected. This is a first step towards the introduction of the penicillin biosynthesis pathway in H. polymorpha.

  3. Diacylglycerol Acyltransferase-1 Localizes Hepatitis C Virus NS5A Protein to Lipid Droplets and Enhances NS5A Interaction with the Viral Capsid Core*

    Science.gov (United States)

    Camus, Gregory; Herker, Eva; Modi, Ankit A.; Haas, Joel T.; Ramage, Holly R.; Farese, Robert V.; Ott, Melanie

    2013-01-01

    The triglyceride-synthesizing enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) plays a critical role in hepatitis C virus (HCV) infection by recruiting the HCV capsid protein core onto the surface of cellular lipid droplets (LDs). Here we find a new interaction between the non-structural protein NS5A and DGAT1 and show that the trafficking of NS5A to LDs depends on DGAT1 activity. DGAT1 forms a complex with NS5A and core and facilitates the interaction between both viral proteins. A catalytically inactive mutant of DGAT1 (H426A) blocks the localization of NS5A, but not core, to LDs in a dominant-negative manner and impairs the release of infectious viral particles, underscoring the importance of DGAT1-mediated translocation of NS5A to LDs in viral particle production. We propose a model whereby DGAT1 serves as a cellular hub for HCV core and NS5A proteins, guiding both onto the surface of the same subset of LDs, those generated by DGAT1. These results highlight the critical role of DGAT1 as a host factor for HCV infection and as a potential drug target for antiviral therapy. PMID:23420847

  4. Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

    Science.gov (United States)

    Knutzon, Deborah S.; Hayes, Thomas R.; Wyrick, Annette; Xiong, Hui; Maelor Davies, H.; Voelker, Toni A.

    1999-01-01

    Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels. PMID:10398708

  5. Polymorphism of rs1044925 in the acyl-CoA:cholesterol acyltransferase-1 gene and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

    Directory of Open Access Journals (Sweden)

    Yan Ting-Ting

    2010-12-01

    Full Text Available Abstract Background The association of rs1044925 polymorphism in the acyl-CoA:cholesterol acyltransferase-1 (ACAT-1 gene and serum lipid profiles is not well known in different ethnic groups. Bai Ku Yao is a special subgroup of the Yao minority in China. The present study was carried out to clarify the association of rs1044925 polymorphism in the ACAT-1 gene and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 626 subjects of Bai Ku Yao and 624 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples. Genotyping of rs1044925 polymorphism in the ACAT-1 gene was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Results The levels of serum total cholesterol (TC, high-density lipoprotein cholesterol (HDL-C, apolipoprotein (Apo AI and ApoB were lower in Bai Ku Yao than in Han (P P P P P P Conclusions These results suggest that the polymorphism of rs1044925 in the ACAT-1 gene is mainly associated with female serum TC, LDL-C and ApoB levels in the Bai Ku Yao population. The C allele carriers had lower serum TC, LDL-C and ApoB levels than the C allele noncarriers.

  6. Rosiglitazone inhibits expression of acyl-coenzyme A:cholesterol acyltransferase-1 in THP-1 macrophages induced by advanced glycation end-products

    Institute of Scientific and Technical Information of China (English)

    Yang Qihong; Xu Qiang; Zhang Hong; Si Liangyi

    2008-01-01

    Objective: To investigate the effects of rosiglitazone, a synthetic ligand of peroxisome proliferators-activated receptor gamma (PPARγ), on the expression of acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in phorbol myristate acetate (PMA)-pretreated THP-1 cells after the inducement of advanced glycation end products (AGEs). Methods: After THP-1 cells were cultured in the presence of 0.1 umol/L PMA for 72 h to induce phagocytic differentiation, the obtained THP-1 macrophages were treated with rosiglitazone for 4 h at different concentrations (1,5 or 10 μmol/L) and then exposed to AGEs-modified bovine serum albumin (AGEs-BSA) for 24 h at a concentration of 200 mg/L. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis were performed to detect the mRNA and protein expressions of ACAT-1 respectively. Results: Administration of AGEs-BSA (200 mg/L) into the THP-1 macrophages resulted in up-regulation of ACAT-1 at mRNA and protein levels when compared with the expressions in macrophages incubated with serum-free RPM11640. Pretreatment of rosiglitazone inhibited significantly the increased expression of ACAT-1 induced by AGEs-BSA in a concentration-dependent manner. Conclusion: PPARγ activation by rosiglitazone down-regulates ACAT-1 expression induced by AGEs in THP-1 macrophages, which might provide a new way for treating atherogenesis in diabetic patients.

  7. Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

    Science.gov (United States)

    Yamaoka, Yasuyo; Achard, Dorine; Jang, Sunghoon; Legéret, Bertrand; Kamisuki, Shogo; Ko, Donghwi; Schulz-Raffelt, Miriam; Kim, Yeongho; Song, Won-Yong; Nishida, Ikuo; Li-Beisson, Yonghua; Lee, Youngsook

    2016-11-01

    Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

  8. Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

    Science.gov (United States)

    Yu, Kyung Ok; Jung, Ju; Kim, Seung Wook; Park, Chul Hwan; Han, Sung Ok

    2012-01-01

    The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol.

  9. The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinus communis: cloning and molecular characterization of two LPAT genes that are expressed in castor seeds.

    Science.gov (United States)

    Arroyo-Caro, José María; Chileh, Tarik; Kazachkov, Michael; Zou, Jitao; Alonso, Diego López; García-Maroto, Federico

    2013-02-01

    The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol.

  10. Molecular Cloning and Characterization of a Novel Human Glycine-N-acyltransferase Gene GLYATL1, Which Activates Transcriptional Activity of HSE Pathway

    Directory of Open Access Journals (Sweden)

    Long Yu

    2007-05-01

    Full Text Available The glycine-N-acyltransferase (GLYAT is well known to be involved in thedetoxification of endogenous and exogenous xenobiotic acyl-CoA's in mammals.Unfortunately, the knowledge about the gene encoding GLYAT is very limited. Here wereport a novel gene encoding a GLYAT member, designated as GLYATL1, which was1546 base pairs in length and contained an open reading frame (ORF encoding apolypeptide of 302 amino acids. GLYATL1 was a split gene that was consisted of 7 exonsand 6 introns and mapped to chromosome 11q12.1. The expression of GLYATL1 could befound in liver, kidney, pancreas, testis, ovary and stomach among 18 human tissues by RT-PCR analysis. Subcellular localization of myc-tagged GLYATL1 fusion protein revealedthat GLYATL1 was distributed primarily in the cytoplasm of COS-7 cells. Furthermore,through the pathway profiling assay, the GLYATL1 protein was found to activate HSEsignaling pathway in a dose-dependent manner when overexpressed in HEK293T cells.

  11. Glycerol-3-Phosphate Acyltransferase 6 (GPAT6) Is Important for Tapetum Development in Arabidopsis and Plays Multiple Roles in Plant Fertility

    Institute of Scientific and Technical Information of China (English)

    Xiao-Chuan Li; Jun Zhu; Jun Yang; Guo-RuiZhang; Wei-Feng Xing; Sen Zhang; Zhong-NanYang

    2012-01-01

    Glycerol-3-phosphate acyltransferase (GPAT) mediates the initial synthetic step for the formation of glycerolipids,which act as the major components of biological membranes and the principal stored forms of energy.GPAT6 is a member of the Arabidopsis GPAT family,which is crucial for cutin biosynthesis in sepals and petals.In this work,a functional analysis of GPAT6 in anther development and plant fertility was performed.GPAT6 was highly expressed in the tapetum and microspores during anther development.The knockout mutant,gpat6,caused a massive reduction in seed production.This report shows that the ablation of GPAT6 caused defective tapetum development with reduced endoplasmic reticulum (ER) profiles in the tapetum,which largely led to the abortion of pollen grains and defective pollen wall formation.In addition,pollen germination and pollen tube elongation were affected in the mutant plants.Furthermore,the double mutant analysis showed that GPAT6 and GPAT1 make joint effects on the release of microspores from tetrads and stamen filament elongation.This work shows that GPAT6 plays multiple roles in stamen development and fertility in Arabidopsis.

  12. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation.

    Science.gov (United States)

    Yu, Jung Hwan; Lee, Yoo Jeong; Kim, Hyo Jung; Choi, Hyeonjin; Choi, Yoonjeong; Seok, Jo Woon; Kim, Jae-woo

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans.

  13. Structural analysis of the alcohol acyltransferase protein family from Cucumis melo shows that enzyme activity depends on an essential solvent channel.

    Science.gov (United States)

    Galaz, Sebastián; Morales-Quintana, Luis; Moya-León, María Alejandra; Herrera, Raúl

    2013-03-01

    Alcohol acyltransferases (AAT) play a key role in ester biosynthesis. In Cucumis melo var. cantalupensis, AATs are encoded by a gene family of four members (CmAAT1-4). CmAAT1, CmAAT3 and CmAAT4 are capable of synthesizing esters, with CmAAT1 the most active. CmAAT2 is inactive and has an Ala268 residue instead of a threonine which is present in all other active AATs, although the role of this residue is still unclear. The present work aims to understand the molecular mechanism involved in ester biosynthesis in melon fruit and to clarify the importance of the Ala268 residue. First, structural models for each protein were built by comparative modelling methodology. Afterwards, conformational interaction between the protein and several ligands, alcohols and acyl-CoAs was explored by molecular docking and molecular dynamics simulation. Structural analysis showed that CmAATs share a similar structure. Also, well-defined solvent channels were described in the CmAATs except for CmAAT2 which does not have a proper channel and instead has a small pocket around Ala268. Residues of the catalytic HxxxD motif interact with substrates within the solvent channel, with Ser363 also important. Strong binding interaction energies were described for the best substrate couple of each CmAAT (hexyl-, benzyl- and cinnamyl-acetate for CmAAT1, 3 and 4 respectively). CmAAT1 and CmAAT2 protein surfaces share similar electrostatic potentials; nevertheless the entrance channels for the substrates differ in location and electrostatic character, suggesting that Ala268 might be responsible for that. This could partly explain the major differences in activity reported for these two enzymes.

  14. A Salmonella typhimurium-translocated Glycerophospholipid:Cholesterol Acyltransferase Promotes Virulence by Binding to the RhoA Protein Switch Regions

    Energy Technology Data Exchange (ETDEWEB)

    LaRock, Doris L.; Brzovic, Peter S.; Levin, Itay; Blanc, Marie-Pierre; Miller, Samuel I.

    2012-08-24

    Salmonella enterica serovar typhimurium translocates a glycerophospholipid: cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.

  15. Iridoid enriched fraction from Ajuga iva reduce cholesterolemia, triacylglycerolemia and increase the lecithin:cholesterol acyltransferase activity of rats fed a cholesterol-rich diet

    Directory of Open Access Journals (Sweden)

    Marie A. Lacaille-Dubois

    2012-02-01

    Full Text Available Objective: In this study, we examined the effect of iridoid (I derived from lyophilized aqueous extract of Ajuga iva on serum HDL2 and HDL3 compositions and lecithin:cholesterol acyltransferase (LCAT activity, enzyme responsible for reverse cholesterol transport. Methods: Male Wistar rats (n=24 weighing 120±5 g were fed a diet containing 1% cholesterol-rich diet for 15 days. After this phase, the hypercholesterolemic (HC rats were divided into groups fed the same diet and received or not doses (5, 10 or 15 mg/kg b.w by intraperitoneal injection of iridoid for 15 days. Results: Compared to HC group, serum total cholesterol value was 1.4- and 1.2-fold lower in the I5-HC and I10-HC groups. C-HDL2 and C-HDL3 values were increased in the I5-HC, I10-HC and I15-HC groups (3.2- and 4-, 2.2- and 4.2-, and 3.2- and 8.7-fold, respectively. HDL2 amounts were 4-, 4- and 2.5-fold higher in the I5-HC, I10-HC and I15-HC groups. In HDL3, phospholipids contents were similar, whereas, unesterified cholesterol values were 3.3-, 2.8- and 3-fold higher in the I5-HC, I10-HC and I15-HC groups. In HDL2, cholesteryl esters contents were significantly higher in the groups treated with iridoid (p<0.05. LCAT activity was increased in the I5-HC and I10-HC groups. Conclusion: Treatment with iridoid at doses 5 or 10mg/kg b.w reduce cholesterolemia. These molecules act efficiently on the efflux of cholesterol from peripheral tissues to the liver by increasing LCAT activity. [J Exp Integr Med 2012; 2(1.000: 55-60

  16. An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans.

    Science.gov (United States)

    Roesler, Keith; Shen, Bo; Bermudez, Ericka; Li, Changjiang; Hunt, Joanne; Damude, Howard G; Ripp, Kevin G; Everard, John D; Booth, John R; Castaneda, Leandro; Feng, Lizhi; Meyer, Knut

    2016-06-01

    Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 µm Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wild-type DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans.

  17. Site-directed mutagenesis from Arg195 to His of a microalgal chloroplastidial glycerol-3-phosphate acyltransferase causes an increase in phospholipid levels in yeast

    Directory of Open Access Journals (Sweden)

    Long-Ling eOuyang

    2016-03-01

    Full Text Available To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT to the first acylation of glycerol-3-phosphate (G-3-P, the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT and the subcellular localization of the encoded protein LiGPAT. A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. LiGPAT was exclusively localized to chloroplasts, which was shown by co-expression of LiGPAT with eGFP in Chlamydomonas reinhardtii and by immunogold labeling in L. incisa. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT, we established the heterologous expression of either mLiGPAT or its mutant (Arg195His (sdmLiGPAT in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest.

  18. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

    Science.gov (United States)

    Rom, Oren; Jeries, Helana; Hayek, Tony; Aviram, Michael

    2017-01-02

    During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017.

  19. Glycerol-3-phosphate acyltransferase-4-deficient mice are protected from diet-induced insulin resistance by the enhanced association of mTOR and rictor.

    Science.gov (United States)

    Zhang, Chongben; Cooper, Daniel E; Grevengoed, Trisha J; Li, Lei O; Klett, Eric L; Eaton, James M; Harris, Thurl E; Coleman, Rosalind A

    2014-08-01

    Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser⁴⁷³) and Akt(Thr³⁰⁸). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance.

  20. Application of a newly identified and characterized 18-o-acyltransferase in chemoenzymatic synthesis of selected natural and nonnatural bioactive derivatives of phoslactomycins.

    Science.gov (United States)

    Ghatge, Mohini S; Palaniappan, Nadaraj; Alhamadsheh, Ma'moun M; DiBari, Jessica; Reynolds, Kevin A

    2009-06-01

    Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS(3) from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS(3) open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (k(cat) of 28 +/- 3 min(-1) and K(m) of 88 +/- 16 microM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS(3) was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.

  1. Monoacylglycerol O-acyltransferase 1 is regulated by peroxisome proliferator-activated receptor γ in human hepatocytes and increases lipid accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Jung Hwan [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Lee, Yoo Jeong [Division of Metabolic Disease, Center for Biomedical Sciences, National Institutes of Health, Cheongwon-gun, Chungbuk 363-951 (Korea, Republic of); Kim, Hyo Jung; Choi, Hyeonjin [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Choi, Yoonjeong; Seok, Jo Woon [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Kim, Jae-woo, E-mail: japol13@yuhs.ac [Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 120-752 (Korea, Republic of); Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2015-05-08

    Monoacylglycerol O-acyltransferase (MGAT) is an enzyme that is involved in triglyceride synthesis by catalyzing the formation of diacylglycerol from monoacylglycerol and fatty acyl CoAs. Recently, we reported that MGAT1 has a critical role in hepatic TG accumulation and that its suppression ameliorates hepatic steatosis in a mouse model. However, the function of MGAT enzymes in hepatic lipid accumulation has not been investigated in humans. Unlike in rodents, MGAT3 as well as MGAT1 and MGAT2 are present in humans. In this study, we evaluated the differences between MGAT subtypes and their association with peroxisome proliferator-activated receptor γ (PPARγ), a regulator of mouse MGAT1 expression. In human primary hepatocytes, basal expression of MGAT1 was lower than that of MGAT2 or MGAT3, but was strongly induced by PPARγ overexpression. A luciferase assay as well as an electromobility shift assay revealed that human MGAT1 promoter activity is driven by PPARγ by direct binding to at least two regions of the promoter in 293T and HepG2 cells. Moreover, siRNA-mediated suppression of MGAT1 expression significantly attenuated lipid accumulation by PPARγ overexpression in HepG2 cells, as evidenced by oil-red-O staining. These results suggest that human MGAT1 has an important role in fatty liver formation as a target gene of PPARγ, and blocking MGAT1 activity could be an efficient therapeutic way to reduce nonalcoholic fatty liver diseases in humans. - Highlights: • PPARγ promotes MGAT1 expression in human primary hepatocytes. • PPARγ directly regulates MGAT1 promoter activity. • Human MGAT1 promoter has at least two PPARγ-binding elements. • Inhibition of MGAT1 expression attenuates hepatic lipid accumulation in humans.

  2. The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa alphabeta heterodimer in an undissociated form.

    Science.gov (United States)

    Fernández, Francisco J; Cardoza, Rosa E; Montenegro, Eduardo; Velasco, Javier; Gutiérrez, Santiago; Martín, Juan F

    2003-05-01

    The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase alphabeta heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the beta subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself. When the penDE gene of A. nidulans was expressed in P. chrysogenum npe6 and npe8 or in Acremonium chrysogenum, the IAT formed had a molecular mass of 40 kDa. On the other hand, when the penDE gene originating from P. chrysogenum was expressed in A. chrysogenum, the active IAT had a molecular mass of 29 kDa. The intronless form of the penDE gene cloned from an A. nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with antibodies to IAT. This 40-kDa protein remained unprocessed even when treated with A. nidulans crude extract. In contrast, the P. chrysogenum penDE intronless gene cloned from a cDNA library was expressed in E. coli, and the IAT was self-processed efficiently into its alpha (29 kDa) and beta (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self-process their respective proIAT protein and to maintain the alpha and beta subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity.

  3. Effects of the diacylglycerol o-acyltransferase 1 (DGAT1) K232A polymorphism on fatty acid, protein, and mineral composition of dairy cattle milk.

    Science.gov (United States)

    Bovenhuis, H; Visker, M H P W; Poulsen, N A; Sehested, J; van Valenberg, H J F; van Arendonk, J A M; Larsen, L B; Buitenhuis, A J

    2016-04-01

    Several studies have described associations between the diacylglycerol o-acyltransferase 1 (DGAT1) K232A polymorphism and routinely collected milk production traits but not much is known about effects of the DGAT1 polymorphism on detailed milk composition. The aim of this study was to estimate effects of the DGAT1 polymorphism on milk fatty acid, protein, and mineral composition. We looked for effects that were significant and consistent in Danish Holstein Friesian (HF), Danish Jersey, and Dutch HF as these are likely to be true effects of the DGAT1 K232A polymorphism rather than being effects of linked loci. For fatty acid composition, significant and consistent effects of the DGAT1 polymorphism were detected on C14:0, C16:0, C15:0, C16:1, C18:1 cis-9, conjugated linoleic acid (CLA) cis-9,trans-11, C18:2 cis-9,cis-12, and C18:3 cis-9,cis-12,cis-15 content (percent by weight, wt/wt %). For C16:0, C16:1, and C18:1 cis-9, the DGAT1 polymorphism explained more than 10% of the phenotypic variation. Significant effects on milk protein composition in Dutch HF could not be confirmed in Danish Jersey or Danish HF. For mineral content, significant and consistent effects of the DGAT1 polymorphism on calcium, phosphorus, and zinc were detected. In the Dutch HF population, the contribution of the DGAT1 K232A polymorphism to phenotypic variance was 12.0% for calcium, 8.3% for phosphorus, and 6.1% for zinc. Different from effects on fatty acid composition, effects of the DGAT1 polymorphism on yields of long-chain fatty acids C18:1 cis-9, CLA cis-9,trans-11, C18:2 cis-9,cis-12, and C18:3 cis-9,cis-12,cis-15 were not significant. This indicates that effects of DGAT1 on these fatty acids are indirect, not direct, effects: DGAT1 affects de novo synthesis of fatty acids and, consequently, the contribution of the long-chain fatty acids to total fat is decreased. In addition, effects of the DGAT1 polymorphism on yields of Ca, P, and Zn were not significant, which indicates that effects

  4. A novel human ghrelin variant (In1-ghrelin and ghrelin-O-acyltransferase are overexpressed in breast cancer: potential pathophysiological relevance.

    Directory of Open Access Journals (Sweden)

    Manuel D Gahete

    Full Text Available The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex, with Ex1-Ex4 encoding a 117 amino-acid (aa preproprotein that is known to be processed to yield a 28-aa (ghrelin and/or a 23-aa (obestatin mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant, which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001 but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001, ghrelin receptor-type 1b (GHSR1b; p = 0.049 and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009, but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.

  5. Impact of single nucleotide polymorphisms in leptin, leptin receptor, growth hormone receptor, and diacylglycerol acyltransferase (DGAT1) gene loci on milk production, feed, and body energy traits of UK dairy cows.

    Science.gov (United States)

    Banos, G; Woolliams, J A; Woodward, B W; Forbes, A B; Coffey, M P

    2008-08-01

    The impact of 9 single nucleotide polymorphisms (SNP) in the leptin (LEP), leptin receptor (LEPR), growth hormone receptor (GHR), and diacylglycerol acyltransferase (DGAT1) gene loci on daily milk production, feed intake, and feed conversion, and weekly measures of live weight, BCS, and body energy traits was evaluated using genetic and phenotypic data on 571 Holstein cows raised at the Langhill Dairy Cattle Research Center in Scotland. Six SNP were typed on the LEP gene and 1 on each of the other 3 loci. Of the 6 LEP SNP, 3 were in very high linkage disequilibrium, meaning there is little gain in typing all of them in the future. Seven LEP haplotypes were identified by parsimony-based analyses. Random-regression allele-substitution models were used to assess the impact of each SNP allele or haplotype on the traits of interest. Diacylglycerol acyltransferase had a significant effect on milk yield, whereas GHR significantly affected feed intake, feed conversion, and body energy traits. There was also evidence of dominance in allelic effects on milk yield and BCS. The LEP haplotype CCGTTT (corresponding to leptin SNP C207T, C528T, A1457G, C963T, A252T, and C305T, respectively) significantly affected milk yield and feed and dry matter intake. Animals carrying this haplotype produced 3.13 kg more milk daily and consumed 4.64 kg more feed. Furthermore, they tended to preserve more energy than average. Such results may be used to facilitate genetic selection in animal breeding programs.

  6. A second gene for acyl-(acyl-carrier-protein): glycerol-3-phosphate acyltransferase in squash, Cucurbita moschata cv. Shirogikuza(*), codes for an oleate-selective isozyme: molecular cloning and protein purification studies.

    Science.gov (United States)

    Nishida, I; Sugiura, M; Enju, A; Nakamura, M

    2000-12-01

    A new isogene for acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) in squash has been cloned and the gene product was identified as oleate-selective GPAT. Using PCR primers that could hybridise with exons for a previously cloned squash GPAT, we obtained two PCR products of different size: one coded for a previously cloned squash GPAT corresponding to non-selective isoforms AT2 and AT3, and the other for a new isozyme, probably the oleate-selective isoform AT1. Full-length amino acid sequences of respective isozymes were deduced from the nucleotide sequences of genomic genes and cDNAs, which were cloned by a series of PCR-based methods. Thus, we designated the new gene CmATS1;1 and the other one CmATS1;2. Genome blot analysis revealed that the squash genome contained the two isogenes at non-allelic loci. AT1-active fractions were partially purified, and three polypeptide bands were identified as being AT1 polypeptides, which exhibited relative molecular masses of 39.5-40.5 kDa, pI values of 6.75-7.15, and oleate selectivity over palmitate. Partial amino-terminal sequences obtained from two of these bands verified that the new isogene codes for AT1 polypeptides.

  7. Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals.

    Science.gov (United States)

    Kroon, Johan T M; Wei, Wenxue; Simon, William J; Slabas, Antoni R

    2006-12-01

    Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.

  8. 花生溶血磷脂酸酰基转移酶基因的克隆与表达分析%Cloning and Expression Analysis of Lysophosphatidic Acid Acyltransferase (LPAT) Encoding Gene in Peanut

    Institute of Scientific and Technical Information of China (English)

    陈四龙; 黄家权; 雷永; 任小平; 文奇根; 陈玉宁; 姜慧芳; 晏立英; 廖伯寿

    2012-01-01

    Lysophosphatidic acid acyltransferase (LPAT) is a key enzyme in biosynthesis pathway of vegetable oil in plant. It is important for oil crops to improve oil quality and increase seed oil content through genetic engineering. We constructed a full-length cDNA library of peanut (Arachis hypogaea) seed via a large number of sequences of expressed sequence tag (EST) and gene functional annotation, a lysophosphatidic acid acyltransferase gene, designated AhLPAT, and its genomic DNA sequence were isolated from peanut. The sequence of AhLPAT cDN A was 1 629 bp, and its genomic sequence was 5 331 bp. Bioinformatic analysis showed that AhLPAT was composed of 11 exons and 10 introns with typical GT-AG characteristic in comparison of its sequences of genomic DNA and cDNA by Splign in NCBI. A peptide of 387 amino acid residues with protein molecular weight of 43.2 kD and isoelectric point (p7) of 9.42 were deduced from AhLPAT. Conserved domains prediction indicated that AhLPAT comprised a typical conserved acyltransferase domain and a conserved lysophospholipid acyltransferases domain. The deduced amino acid had a high identity with the LPAT proteins reported from other species. Amino acid similarities of LPAT protein be tween peanut and Tropaeolum majus, Brassica napus, Crambe hispanica subsp. Abyssinica, Ricinus communis, and Arabidopsis thaliana were 90%, 89%, 89%, 88%, and 87%, respectively. A phylogenetic tree was constructed by the Neighbor-Joining method using MEGA5.0. The phylogenetic tree suggested that AhLPAT and AtLPAT2 derived from Arabidopsis thaliana were grouped into the same class. Both AhLPAT and AtLPAT2 were endoplasmic reticulum type LPATs. The tissue specific expression analysis by using quantitative RT-PCR assays indicated that AhLPAT was ubiquitously expressed in root, stem, leaf, flower, gynophore, seed of peanut with the highest level in gynophore and seed. The expression level reached a peak in the stage from 50 to 60 days after flowering. The

  9. Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

    Science.gov (United States)

    Sato, Kenjiro; Takahagi, Hiroki; Kubo, Osamu; Hidaka, Kousuke; Yoshikawa, Takeshi; Kamaura, Masahiro; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Maki, Toshiyuki; Take, Kazumi; Takekawa, Shiro; Kitazaki, Tomoyuki; Maekawa, Tsuyoshi

    2015-08-01

    Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts.

  10. Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape

    Directory of Open Access Journals (Sweden)

    Mosimann Steven C

    2006-12-01

    Full Text Available Abstract Background Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20 catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1, we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1–116His6, with calculated molecular mass of 13,278 Da. Results BnDGAT1(1–116His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1–116His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisΔ13-CoA over oleoyl (18:1cisΔ9-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1–116His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1–116His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. Conclusion Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER.

  11. 莱茵衣藻磷脂二脂酰甘油酰基转移酶3在三酰甘油合成中的功能研究%THE ROLE OF PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE IN BIOSYNTHESIS OF TRIACYLGLYCEROL BY CHLAMYDOMONAS REINHARDTII

    Institute of Scientific and Technical Information of China (English)

    邓晓东; 蔡佳佳; 费小雯

    2014-01-01

    Currently, production of biodiesel by microalgae has been regarded as a promising source of renewable en-ergy. However, the understanding of oil biosynthesis mechanisms in micro-algae is limited. Phospholipid:diacylglycerol acyltransferase catalyzes phospholipid and diacylglycerol to produce triglyceride, a key reaction in triglyceride synthe-sis. In this study, we cloned a fragment of Phospholipid:diacylglycerol acyltransferase homologous gene 3 CrPDAT3 in Chlamydomonas, which was then used to construct a CrPDAT3 RNAi interference vector and transferred into Chlamy-domonas. The results showed that the growth rate of transgenic algae strain was declined. At the same time, the oil con-tent was decreased by 14.65%-45.15%, showing that the CrPDAT3 playing an important role in oil biosynthesis.%为研究磷脂二脂酰甘油酰基转移酶(PDAT)在三酰甘油合成中的功能,克隆了莱茵衣藻(Chlamydomonas reinhardtii) PDAT同源基因CrPDAT3干涉片段,通过构建CrPDAT3 RNAi 干涉载体并转化莱茵衣藻,对 CrPDAT3基因有效沉默,结果显示转基因藻株生长减缓,油脂含量下降14.65%-45.15%,说明CrPDAT3对油脂合成起到重要的作用。研究结果对于该基因应用于微藻油脂的遗传改良将起到重要作用。

  12. Cloning and Characterization of Diacylglycerol Acyltransferase Gene (NtDGAT2) from Tobacco (Nicotiana tabacum L.)%烟草二脂酰甘油酰基转移酶基因(NtDGAT2)的克隆与功能分析

    Institute of Scientific and Technical Information of China (English)

    阳天泉; 徐荣华; 刘爱忠

    2013-01-01

    Diacylglycerol acyltransferase (DGAT2), a key enzyme for lipid biosynthesis, plays a critical role in oil accumulation in oilseeds. In this study, using the silico cloning technique combined with RT-PCR method, a novel DGAT2 gene with the length of 999 bp encoding 332 amino acids was isolated from seed cDNAs of tobacco (Nicotiana tabacum), named NtDGAT2 (GenBank No JX843807). The sequence analyses showed that the amino acid sequence of NtDGAT2 with the typical functional motifs of DGAT2 was high similarity to those identified from other species. Based on Real-time quantitative PCR expression analysis, NtDGAT2 was expressed in all tissues such as root, stem, leaf, flower and developing seed; in particular, NtDGAT2 was highly expressed in flowers and developing seeds. Further, the function of NtDGAT2 was confirmed by heterologous transformation and the functional complementary experiments in yeast.%二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT2)是植物储存油脂生物合成过程中的关键酶,对种子储存油脂累积具有重要的生理作用.本文采用电子克隆与实验相结合的方法,从烟草种子cDNA中克隆到DGAT2基因的开放阅读框序列,命名为NtDGAT2 (GenBank登录号JX843807),其序列长999 bp,编码332个氨基酸.多序列比对和进化分析表明该基因编码蛋白与其他植物DGAT2具有较高相似性和典型的DGAT2结构域.利用Real-time PCR定量表达分析显示Nt-DGA T2在烟草种子、花、茎、叶和根里面都有表达,且在发育中的种子和花的发育过程大量表达.酵母互补实验证实该基因编码蛋白具有DGAT酶活性.

  13. Cloning and Characterization of Phospholipids:Diacylglycerol Acyltransferase (BnPDAT1) cDNA from Brassica napus L.%甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1)cDNA的克隆和功能鉴定

    Institute of Scientific and Technical Information of China (English)

    谭太龙; 冯韬; 罗海燕; 彭烨; 刘睿洋; 官春云

    2016-01-01

    Phospholipids:diacylglycerol acyltransferase (PDAT1) is a key enzyme in triacylglycerol (TAG) biosynthesis of plants. In this study, three novel PDAT1 coding sequences (CDSs) were isolated from cDNA of Brassica napus L. cv. Xiangyou 15 seeds, which were mapped to the chromosomes A02, A10, and C09, and designated as BnPDAT1-A02,BnPDAT1-A10, and BnPDAT1-C09, respectively. Three BnPDAT1 CDSs were 1998, 2002, and 2005 bp in length and encoded predicted proteins with 665, 666, and 667 amino acid residues, respectively. BnPDAT1 proteins were predicted to be located on the cell membrane and have a typical PDAT1 conserved domain. Multiple sequence alignments and phylogenetic analysis showed that the deduced amino acid sequences of BnPDAT1 were highly homologous to previously reported PDAT1 inBrassica oleracea,Arabidopsis thalian, and Eruca sativa. Furthermore, the catalytic enzyme activity of the cloned BnPDAT1 genes was confirmed by the yeast comple-mentary experiment. The expression level of BnPDAT1s increased gradually in seed development and reached the maximum from 25 to 30 days after flowering. However, three BnPDAT1 copies were also found to be different in expression pattern.%磷脂二酰甘油酰基转移酶(phospholipids:diacylglycerol acyltransferase,PDAT1)是植物三酰甘油(triacylgly-cerol,TAG)合成的关键酶.本文在甘蓝型油菜湘油15号cDNA中克隆到3个PDAT1全长编码序列(coding sequence,CDS),经比对分别定位于A02、A10、C09染色体,分别命名为BnPDAT1-A02、BnPDAT1-A10和BnPDAT1-C09,其序列长分别为1998、2002和2005 bp,各自编码665、666、667个氨基酸.预测BnPDAT1基因编码蛋白定位于细胞质膜,具有典型的PDAT1保守结构域.多序列比对和进化分析表明,BnPDAT1基因编码蛋白与甘蓝、拟南芥、亚麻芥PDAT1蛋白具有较高的同源性.酵母互补实验证实,该基因编码蛋白具有PDAT1酶活性.BnPDAT1基因在湘油15号中的表达现先上升后降低趋势,在开花后25

  14. 缺血性脑血管病患者血浆卵磷脂-胆固醇酰基转移酶活性与其脂质代谢%Activity of plasma lecithin cholesterol acyltransferase and its lipid metabolism in patients with ischemic cerebrovascular disease

    Institute of Scientific and Technical Information of China (English)

    初开秋; 唐晓燕; 田清武; 任立晟; 张少燕

    2005-01-01

    酰基转移酶活性分别与高密度脂蛋白胆固醇及载脂蛋白A1呈正相关(r=0.247,P<0.05;r=0.303,P<0.01),而与低密度脂蛋白胆固醇和红细胞膜胆固醇呈负相关(r=-0.212,P<0.05;r=-0.346,P<0.01).结论:缺血性脑血管病患者血浆卵磷脂-胆固醇酰基转移酶活性下降,且并非继发于脑梗死发生后,其活性变化与高密度脂蛋白胆固醇及载脂蛋白A1呈正相关,与低密度脂蛋白胆固醇及红细胞膜胆固醇呈负相关性.%BACKGROUND: Abnormal lipid metabolism is one of the risk factors in patients with ischemic cerebral disorders, and is correlated with the changes of lecithin cholesterol acyltransferase activity.OBJECTIVE: To observe the relationship between the changes of lecithin cholesterol acyltransferase activity and lipid content in red blood cell membrane.DESIGN: A case-control study(experimental group with control as standard level).SETTING: Department of clinical laboratory, emergency room and department of neurology of a hospital affiliated to a medical college of a university.PARTICIPANTS: Totally 105 inpatients and outpatients with cerebrovascular diseases were selected from the Department of Neurology, Affiliated Hospital of Medical College of Qingdao University, from March 2002 to December 2003. They accorded with the Diagnostic Criteria set at the Second National Conference on Cerebrovascular Diseases. A total of 42 patients with cerebral arteriosclerosis and 63 patients with cerebral infarction were selected as patients group consisting of 67 males and 38 females. Another 65 healthy people receiving physical examination in the hospital, 36 males and 29 females, were selected as control group.METHODS: Venous blood of 8 mL was drawn from the participants on an empty stomach. We assayed the activity of lecithin cholesterol acyltransferase,high density lipoprotein cholesterol, low density lipoprotein cholesterol,apolipoprotein A1 and apolipoprotein B. Red blood cell membrane

  15. Plasma Atherogenic Index and Acyl-coenzyme A Cholesterol Acyltransferase 2 of Obese Adolescents after Weight Reduction%4周运动减肥对肥胖青少年血浆致动脉粥样硬化指数和脂酰辅酶A胆固醇酰基转移酶2的影响

    Institute of Scientific and Technical Information of China (English)

    林云; 陈文鹤

    2012-01-01

    Objective To observe the effect of weight reduction on plasma atherogenic index and cyl-coenzyme A cholesterol acyltransferase 2 (ACAT2) of obese adolescents. Methods 30 obese adolescents took part in 4-week aerobic exercise and diet program for weight reduction. Results After 4 weeks of exercise and diet,the body weight,fat percentage,BMI and serum insulin of the subjects reduced significantly(P < 0.01);the level of LPL and the ratio of HDL-C/LDL-C increased and the level of TG, TC,HDL-C,LDL-C,atherogenic index and ACAT2 decreased significantly (P < 0.01). Conclusion Weight reduction through exercise and proper diet effectively improves somatometric measurements,lipid metabolism and insulin resistance to a certain extent in obese adolescents.%目的:观察运动减肥对肥胖青少年血浆致动脉粥样硬化指数和脂酰辅酶A胆固醇酰基转移酶等动脉粥样硬化致病相关因子的影响.方法:以参加2011年上海巅峰运动减肥夏令营的30名肥胖青少年为对象,进行4周中小强度有氧运动为主结合适当饮食控制的减肥运动训练.分别于入营第一天和出营前一天进行身体形态及血液指标测试.结果:经过夏令营4周有氧运动训练,肥胖青少年体重、体脂率、BMI与运动前相比显著下降,空腹血清胰岛素、血脂水平与运动前相比均明显改善,血浆致动脉粥样硬化指数、脂酰辅酶A胆固醇酰基转移酶2水平显著降低.结论:4周有氧运动明显降低了肥胖青少年的肥胖程度和胰岛素水平,改善血脂代谢,在一定程度上降低了动脉粥样硬化发病的风险.

  16. 小桐子甘油-3-磷酸酰基转移酶(JcGPAT)cDNA的克隆与序列分析%Cloning and Sequence Analysis of sn-Glycerol-3-Phosphate Acyltransferase Gene (JcGPAT) from Jatropha curcas L.

    Institute of Scientific and Technical Information of China (English)

    张楠; 徐荣华; 刘小烛; 刘爱忠

    2011-01-01

    甘油-3-磷酸酰基转移酶是植物生物合成储存油脂过程中的关键酶,对油料作物种子含油量具有重要的限制作用.本研究以植物甘油-3-磷酸酰基转移酶同源基因的保守区域序列为基础,设计简并引物,结合RACE技术,从能源植物小桐子种子中克隆获得JcGPAT基因的cDNA全长序列(GenBank登录号HQ395225).JcGPATcDNA核苷酸序列长度为1 672 bp,开放阅读框为1125 bp,编码375个氨基酸.该基因具有明显的GPAT基因结构域,其编码的氨基酸序列与油桐、蓖麻等植物具有很高的同源性.RT-PCR表达分析表明,该基因在小桐子发育的种子、叶、根尖等多个组织表达.%sn-Glycerol-3-phosphate acyltransferase (GPAT), a key enzyme in lipid metabolism, plays a critical role in biosynthesis of lipids in plants. In this study, based on the conserved regions of GPAT genes available from GenBank database, we designed degenerate primers and obtained the cDNA sequences of GPAT gene by RACE technology from Jatropha curcas, named JcGPAT (GenBank accession no. HQ395225). The full length cDNA is 1 672 bp, encoding 375 amino acids which have a high identity (ranging from 78% to 95%) with GPAT genes in other plants reported such as castor (Ricinus communis) and tung tree (Vernicia fordii). RT-PCR analysis showed that JcGPAT was expressed in different tissues including the developing seeds, leaf, root tip and callus.

  17. Seed-Specific Over-Expression of a Diacylglycerol Acyltransferase 1 Gene (VgDGAT1) Increase Seed Oil Accumulation in Camelina sativa%种子特异表达二酰甘油酰基转移酶基因(VgDGAT1)提高亚麻荠种子油脂积累

    Institute of Scientific and Technical Information of China (English)

    苑丽霞; 毛雪; 高昌勇; 张莉; 薛金爱; 杨致荣; 李润植

    2015-01-01

    Camelina sativa is an important non-food industrial oilseed with“low-input, high efifciency and en-environment- friendly”. To enhance seed oil content for meeting an increasing market demand, a cDNA clone (Vg- DGAT1) encoding type 1 diacylglycerol acyltransferase from Vernonia galamensis with the higher DGAT enzymatic activity was introduced into camelina by Agrobacterium-mediated floral dip infiltration. Seed-specific over-expression of VgDGAT1 significantly enhanced the DGAT activity in the transgenic seeds by 30 folds more compared to the wild-type control, resulting in seed oil content increase from 37% (dry weight) in the wild-type seeds up to 46%–51% in the transgenics. However, seed protein level was not significant difference between the transgenic and the wild type, indicating that genetic modification on DGAT can break up the negative genetic linkage of oil and protein contents in seeds. Moreover, the seed-specific high expression of Vg- DGAT1 did not adversely affect seed weight, seed germination and other agronomic traits. Such novel engineered camelina lines with higher seed oil content and no reduction of protein accumulation could be used for breeding new camelina varieties with multiple excellent agronomic traits for commercialization.%亚麻荠(Camelina sativa)是一种“低耗、高效、环保”的非粮型工业油料作物。为提高亚麻荠种子含油量,将来源于一种菊科野生油料植物(Vernonia galamensis)高酶活性的二酰甘油酰基转移酶1 cDNA克隆(VgDGAT1)在发育种子中特异表达。VgDGAT1超表达导致转基因亚麻荠种子中DGAT酶活性提高了30多倍。VgDGAT1高表达的亚麻荠种子含油量从野生型的37%提高到46%~51%,而且蛋白质积累未减少,表明对DGAT酶基因进行遗传修饰可打破种子油和蛋白含量的负相关连锁。此外, VgDGAT1高表达也没有对种子重量和种子萌发等农艺性状造成不良影响。这种高油亚麻荠基因工程新

  18. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    Science.gov (United States)

    2015-10-01

    Nat. Chem. Biol. 2, 584−590. (4) Roth , A. F., Wan, J., Bailey, A. O., Sun, B., Kuchar, J. A., Green, W. N., Phinney, B. S., Yates, J. R., 3rd, and...Davis, N. G. (2006) Global analysis of protein palmitoylation in yeast. Cell 125, 1003−1013. (5) Wan, J., Roth , A. F., Bailey, A. O., and Davis, N. G...cerevisiae. J. Biol. Chem. 277, 41268−41273. (14) Roth , A. F., Feng, Y., Chen, L., and Davis, N. G. (2002) The yeast DHHC cysteine-rich domain protein

  19. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosis pathogenesis.

    Science.gov (United States)

    Shi, Shuangping; Ehrt, Sabine

    2006-01-01

    Mycobacterium tuberculosis has evolved to persist in host macrophages, where it faces a nutrient-poor environment and is exposed to oxidative and nitrosative stress. To defend itself against oxidative/nitrosative stress, M. tuberculosis expresses an NADH-dependent peroxidase and peroxynitrite reductase that is encoded by ahpC, ahpD, lpd, and dlaT. In addition to its central role in the peroxynitrite reductase complex, dlaT (Rv2215) also encodes the E2 component of pyruvate dehydrogenase. Here we demonstrate that inactivation of dlaT in the chromosome of H37Rv resulted in a mutant (H37RvDeltadlaT) that displayed phenotypes associated with DlaT's role in metabolism and in defense against nitrosative stress. The H37RvDeltadlaT strain showed retarded growth in vitro and was highly susceptible to killing by acidified sodium nitrite. Mouse macrophages readily killed intracellular H37RvDeltadlaT organisms, and in mice dlaT was required for full virulence.

  20. Identification and characterization of five BAHD acyltransferases involved in hydroxycinnamoyl ester metabolism in chicory

    Directory of Open Access Journals (Sweden)

    Guillaume eLegrand

    2016-06-01

    Full Text Available Chicory (Cichorium intybus accumulates caffeic acid esters with important significance for human health. In this study, we aim at a better understanding of the biochemical pathway of these bioactive compounds. Detailed metabolic analysis reveals that C. intybus predominantly accumulates caftaric and chicoric acids in leaves, whereas isochlorogenic acid (3,5-diCQA was almost exclusively accumulated in roots. Chlorogenic acid (3-CQA was equally distributed in all organs. Interestingly, distribution of the 4 compounds was related to leaf age. Induction with methyljasmonate (MeJA of root cell suspension cultures results in an increase of 3-CQA and 3,5-diCQA contents. Expressed sequence tag libraries were screened using members of the BAHD family identified in arabidopsis and tobacco as baits. The full-length cDNAs of five genes were isolated. Predicted amino acid sequence analyses revealed typical features of BAHD family members. Biochemical characterization of the recombinant proteins expressed in Escherichia coli showed that 2 genes encode HCTs (hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferases, HCT1 and HCT2 whereas 3 genes encode HQTs (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferases, HQT1, HQT2 and HQT3. These results totally agreed with the phylogenetic analysis done with the predicted amino acid sequences. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT3, HCT1 and HCT2 might be more directly associated with CQA accumulation in cell culture in response to MeJA elicitation. Transient expression of HCT1 and HQT1 in tobacco resulted in a higher production of 3-CQA. All together these data confirm the involvement of functionally redundant genes in 3-CQA and related compound synthesis in the Asteraceae family.

  1. Aminocarnitine and acylaminocarnitines: Carnitine acyltransferase inhibitors affecting long-chain fatty acid and glucose metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Clark, D.J.

    1989-01-01

    DL-Aminocarnitine (DL-3-amino-4-trimethylaminobutyrate) and the acylaminocarnitines acetyl-, decanoyl- and palmitoyl-DL-aminocarnitine have been synthesized and tested as inhibitors of carnitine palmitoyl-transferase and carnitine acetyltransferase in vitro and in vivo. Acetyl-DL-aaminocarnitine is the most potent reversible inhibitor of carnitine acetyltransferase reported to date, and is competitive with respect to acetyl-L-carnitine. Mice given acetyl-DL-aminocarnitine metabolize (U-{sup 14}C)acetyl-L-carnitine at about 60% of the rate of control mice. Palmitoyl-DL-aminocarnitine is the most potent reversible inhibitor of carnitine palmitoyltransferase reported to date. Decanoyl-DL-aminocarnitine and DL-aminocarnitine are also very potent inhibitors; all compounds inhibit the catabolism of ({sup 14}C)palmitate to {sup 14}CO{sub 2} in intact mice by at least 50%. Carnitine palmitoyltransferase controls the entry of long-chain fatty acids into the mitochondrial matrix for {beta}-oxidation. The inhibition of carnitine palmitoyltransferase by aminocarnitine or acylaminocarnitines in vivo prevents or reverses ketogenesis in fasted mice, and causes the reversible accumulation of triglycerides in liver, kidney and plasma. Administration of DL-aminocarnitine to streptozotocindiabetic mice lowers plasma glucose levels and improves the glucose tolerance test.

  2. Engineering industrial oil biosynthesis: cloning and characterization of Kennedy pathway acyltransferases from novel oilseed species

    Science.gov (United States)

    For more than twenty years, various industrial, governmental, and academic laboratories have developed and refined genetic engineering strategies aimed at manipulating lipid metabolism in plants and microbes. The goal of these projects is to produce renewable specialized oils that can effectively c...

  3. ACYLTRANSFERASE ACTIVITIES OF THE HIGH-MOLECULAR-MASS ESSENTIAL PENICILLIN-BINDING PROTEINS

    NARCIS (Netherlands)

    ADAM, M; DAMBLON, C; JAMIN, M; ZORZI, W; DUSART, [No Value; GALLENI, M; ELKHARROUBI, A; PIRAS, G; SPRATT, BG; KECK, W; COYETTE, J; GHUYSEN, JM; NGUYENDISTECHE, M; FRERE, JM

    1991-01-01

    The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the

  4. Reprogramming Acyl Carrier Protein Interactions of an Acyl-CoA Promiscuous trans-Acyltransferase

    DEFF Research Database (Denmark)

    Ye, Zhixia; Musiol-Kroll, Ewa Maria; Weber, Tilmann;

    2014-01-01

    on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a noncognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions...... and for engineering modular polyketide synthases to produce analogs....

  5. Data in support of substrate flexibility of a mutated acyltransferase domain and implications for polyketide biosynthesis

    Directory of Open Access Journals (Sweden)

    Stephan Klopries

    2015-12-01

    Full Text Available Enzyme-directed mutasynthesis is an emerging strategy for the targeted derivatization of natural products. Here, data on the synthesis of malonic acid derivatives for feeding studies in Saccharopolyspora erythraea , the mutagenesis of DEBS and bioanalytical data on the experimental investigation of studies on the biosynthetic pathway towards erythromycin are presented.

  6. Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress.

    Science.gov (United States)

    Sirakova, Tatiana D; Dubey, Vinod S; Deb, Chirajyoti; Daniel, Jaiyanth; Korotkova, Tatiana A; Abomoelak, Bassam; Kolattukudy, Pappachan E

    2006-09-01

    Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C(26) was a major fatty acid in this TG, indicating that the TGS1 gene product uses C(26) fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C(26 : 0)-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.

  7. GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor.

    Science.gov (United States)

    Bosson, Régine; Jaquenoud, Malika; Conzelmann, Andreas

    2006-06-01

    The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.

  8. Targeted Expression and Secretion of Human Apoprotein AI,Apoprotein E and Lecithin-choles-terol Acyltransferase from Myogenic Cell

    Institute of Scientific and Technical Information of China (English)

    范乐明; 张慧; 于书真; 陈琪; 魏恩会; 王南; 蔡海江

    2001-01-01

    Objective To investigate the possibility of heterologous expression for apoAI, apoE and LCAT by skeletal muscle cells and secretion into blood and to develop a safe and convenient gene therapy method for atherosclerosis. Methods Viral and nonviral vectors containing apoAI, apoE or LCAT genes were constructed and transfected into myogenic cells in vitro or injected directed into mouse skeletal muscle. The expression efficiencies of these vectors were investigated by ELISA assay for human apoAI and apoE3 and by the proteoliposome method for human LCAT. Genomic DNA was extracted from stable transduced myoblasts and analyzed for the presence of vector sequence by PCR amplifications. Immunocytochemistry assay was also performed to make an intuitionistic detection for the expression of transgene in myoblasts. Results All viral or nonviral vectors constructed in present study expressed the transgenes efficiently in mice skeletal muscles in vivo or cultured myoblasts in vitro. The transgene expression level of cells transfected with AAV-based plasmid vectors were 2-4 times higher then that of cells transfected with conventional plasmid vectors. Additionally, cells transfected with AAV-based bicistronic vector or tricistronic retroviral vector expressed both human apoAI and LCAT simultaneously. The sequences of retroviral or AAV-based plasmid vectors were found to be retained in host cells after transfection when that of conventional plasmid vectors were lost. Furthermore, transduced myoblasts maintained the ability for heterologous expression of human apoAI and LCAT even after differentiation into myotubes. For cells transfected with retroviral vectors, stable transduced clones can be selected by G418 and continued to efficiently express human apoAI and LCAT for 3 months. Conclusion These finds indicated that mice skeletal muscles or cultured myoblasts transduced with viral or non-viral vectors could efficiently express and secret human apoAI, apoE and LCAT. It suggested that the use of nonviral, adenoviral or AAV-based vectors to directly inject into skeletal muscle or the use of polycistronic retroviral to genetically modify myoblasts ex vivo and then implantation back to skeletal muscle to high efficiently and long-term express apoAI, apoE and LCAT in vivo might be a safe and feasible strategy to prevent or reduce the formation of atherosclerotic lesions.

  9. High pre-beta1 HDL concentrations and low lecithin: cholesterol acyltransferase activities are strong positive risk markers for ischemic heart disease and independent of HDL-cholesterol

    DEFF Research Database (Denmark)

    Sethi, Amar A; Sampson, Maureen; Warnick, Russell;

    2010-01-01

    We hypothesized that patients with high HDL-cholesterol (HDL-C) and ischemic heart disease (IHD) may have dysfunctional HDL or unrecognized nonconventional risk factors.......We hypothesized that patients with high HDL-cholesterol (HDL-C) and ischemic heart disease (IHD) may have dysfunctional HDL or unrecognized nonconventional risk factors....

  10. Tigecycline resistance in Acinetobacter baumannii mediated by frameshift mutation in plsC, encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase.

    Science.gov (United States)

    Li, X; Liu, L; Ji, J; Chen, Q; Hua, X; Jiang, Y; Feng, Y; Yu, Y

    2015-03-01

    Acinetobacter baumannii is an important pathogen of healthcare-associated infections and shows multidrug resistance. With the increasing application of tigecycline, isolates resistant to this antibiotic are of growing concern clinically. However, the definitive mechanism of tigecycline resistance remains unclear. To explore the mechanism of tigecycline resistance in A. baumannii, a tigecycline-resistant strain was obtained by increasing the concentration of the antimicrobial in liquid culture. Three mutations were identified by the whole genome comparison, including one synonymous substitution in a hypothetical protein and a frameshift mutation in plsC and omp38. The plsC gene was confirmed to cause decreased susceptibility to tigecycline by a complementation experiment and cellular membrane change was detected by flow cytometry. By measuring the relative growth rate, the fitness cost of plsC was estimated to be approximately 8 %. In conclusion, plsC was found to play an important role in tigecycline resistance in A. baumannii. The minor fitness cost of plsC indicates a high risk of the emergence and development of tigecycline resistance in A. baumannii.

  11. Identification of a single nucleotide polymorphism at intron 16 of the caprine acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) gene.

    Science.gov (United States)

    Angiolillo, Antonella; Amills, Marcel; Urrutia, Baltasar; Doménech, Anna; Sastre, Yolanda; Badaoui, Bouabid; Jordana, Jordi

    2007-02-01

    The DGAT1 gene encodes a microsomal enzyme that catalyses the only committed step in triacylglycerol synthesis by joining diacylglycerol and fatty acyl coenzyme A. In cattle, a K232A substitution in the DGAT1 molecule has a significant effect on enzyme activity and milk fat content. The prominent role of this gene in lipid metabolism led us to undertake the structural characterization of DGAT1 in goats. In this way, we have sequenced a 1552 bp fragment of the goat DGAT1 cDNA, which encompasses most of the coding sequence (from exon 1 to 17), and a genomic fragment covering exons 12 to 17. Multiple alignment of the goat DGAT1 sequences revealed the existence of a single nucleotide polymorphism (SNP) involving a T to C substitution at intron 16. We optimized a primer extension based genotyping method that allowed us to determine that the C variant is a minority allele with frequencies ranging from 0.062 (Murciano-Granadina) to 0.109 (Malagueña). This SNP, although not expected to have any functional effect, might be useful as a genetic marker in association studies to detect additional DGAT1 polymorphisms which might influence fat milk content and other traits of economic interest.

  12. Structural and Functional Investigation of FdhC from Acinetobacter nosocomialis: A Sugar N-Acyltransferase Belonging to the GNAT Superfamily.

    Science.gov (United States)

    Salinger, Ari J; Thoden, James B; Holden, Hazel M

    2016-08-16

    Enzymes belonging to the GNAT superfamily are widely distributed in nature where they play key roles in the transfer of acyl groups from acyl-CoAs to primary amine acceptors. The amine acceptors run the gamut from histones to aminoglycoside antibiotics to small molecules such as serotonin. Whereas those family members that function on histones have been extensively studied, the GNAT enzymes that employ nucleotide-linked sugars as their substrates have not been well characterized. Indeed, though the structures of two of these "amino sugar" GNAT enzymes have been determined within the past 10 years, details concerning their active site architectures have been limited because of a lack of bound nucleotide-linked sugar substrates. Here we describe a combined structural and biochemical analysis of FdhC from Acinetobacter nosocomialis O2. On the basis of bioinformatics, it was postulated that FdhC catalyzes the transfer of a 3-hydroxybutanoyl group from 3-hydroxylbutanoyl-CoA to dTDP-3-amino-3,6-dideoxy-d-galactose, to yield an unusual sugar that is ultimately incorporated into the surface polysaccharides of the bacterium. We present data confirming this activity. In addition, the structures of two ternary complexes of FdhC, in the presence of CoA and either 3-hydroxybutanoylamino-3,6-dideoxy-d-galactose or 3-hydroxybutanoylamino-3,6-dideoxy-d-glucose, were solved by X-ray crystallographic analyses to high resolution. Kinetic parameters were determined, and activity assays demonstrated that FdhC can also utilize acetyl-CoA, 3-methylcrotonyl-CoA, or hexanoyl-CoA as acyl donors, albeit at reduced rates. Site-directed mutagenesis experiments were conducted to probe the catalytic mechanism of FdhC. Taken together, the data presented herein provide significantly new molecular insight into those GNAT superfamily members that function on nucleotide-linked amino sugars.

  13. Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture.

    Science.gov (United States)

    Daniel, Jaiyanth; Deb, Chirajyoti; Dubey, Vinod S; Sirakova, Tatiana D; Abomoelak, Bassam; Morbidoni, Hector R; Kolattukudy, Pappachan E

    2004-08-01

    Mycobacterium tuberculosis enters the host by inhalation of an infectious aerosol and replicates in the alveolar macrophages until the host's immune defense causes bacteriostasis, which leads the pathogen to go into nonreplicative drug-resistant dormancy. The dormant pathogen can survive for decades till the host's immune system is weakened and active tuberculosis develops. Even though fatty acids are thought to be the major energy source required for the persistence phase, the source of fatty acids used is not known. We postulate that the pathogen uses triacylglycerol (TG) as a storage form of fatty acids. Little is known about the biosynthesis of TG in M. tuberculosis. We show that 15 mycobacterial genes that we identified as putative triacylglycerol synthase (tgs) when expressed in Escherichia coli showed TGS activity, and we report some basic catalytic characteristics of the most active enzymes. We show that several tgs genes are induced when the pathogen goes into the nonreplicative drug-resistant state caused by slow withdrawal of O(2) and also by NO treatment, which is known to induce dormancy-associated genes. The gene (Rv3130c) that shows the highest TGS activity when expressed in E. coli shows the highest induction by hypoxia and NO treatment. Biochemical evidence shows that TG synthesis and accumulation occur under both conditions. We conclude that TG may be a form of energy storage for use during long-term dormancy. Therefore, TG synthesis may be an appropriate target for novel antilatency drugs that can prevent the organism from surviving dormancy and thus assist in the control of tuberculosis.

  14. PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor sulfolipid-1.

    Science.gov (United States)

    Kumar, Pawan; Schelle, Michael W; Jain, Madhulika; Lin, Fiona L; Petzold, Christopher J; Leavell, Michael D; Leary, Julie A; Cox, Jeffery S; Bertozzi, Carolyn R

    2007-07-03

    Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL(1278), has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL(1278) and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL(1278) and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2'-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL(1278). Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the Delta papA2 and Delta papA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL(1278)) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.

  15. NCBI nr-aa BLAST: CBRC-CBRI-05-0288 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CBRI-05-0288 ref|YP_512974.1| Acyltransferase [Francisella tularensis subsp. h...olarctica] ref|YP_762841.1| acyltransferase [Francisella tularensis subsp. holarctica OSU18] ref|YP_001427626.1| acyltransferase [Fra...ncisella tularensis subsp. holarctica FTA] emb|CAJ78620.1| Acyltransferase [Francis...ella tularensis subsp. holarctica LVS] gb|ABI82204.1| acyltransferase [Francisell...a tularensis subsp. holarctica OSU18] gb|EBA51928.1| acyltransferase [Francisella tularensis subsp. holarcti

  16. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_624641.1| acyltransferase 3 [Burkholderia cenocepacia AU 1...054] ref|YP_837010.1| acyltransferase 3 [Burkholderia cenocepacia HI2424] ref|ZP_01565190.1| acyltransferase 3 [Burkholder...ia cenocepacia MC0-3] gb|ABF79668.1| acyltransferase 3 [Burkholderia cenocepacia AU 1054] gb|A...BK10117.1| acyltransferase 3 [Burkholderia cenocepacia HI2424] gb|EAV56867.1| acyltransferase 3 [Burkholderia cenocepacia MC0-3] YP_624641.1 1e-65 56% ...

  17. NCBI nr-aa BLAST: CBRC-AGAM-02-0079 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0079 ref|XP_525054.2| PREDICTED: hedgehog acyltransferase isoform 6 [P...an troglodytes] ref|XP_001169314.1| PREDICTED: hedgehog acyltransferase isoform 3 [Pan troglodytes] ref|XP_0...01169359.1| PREDICTED: hedgehog acyltransferase isoform 4 [Pan troglodytes] ref|XP_001169380.1| PREDICTED: hedgehog acyltransferase isoform 5 [Pan troglodytes] XP_525054.2 4e-33 25% ...

  18. Production of medium-chain volatile flavour esters in Pichia pastoris whole-cell biocatalysts with extracellular expression of Saccharomyces cerevisiae acyl-CoA: ethanol O-acyltransferase Eht1 or Eeb1

    DEFF Research Database (Denmark)

    Zhuang, Shiwen; Fu, Junshu; Powell, Chris;

    2015-01-01

    Medium-chain volatile flavour esters are important molecules since they have extensive applications in food, fragrance, cosmetic, paint and coating industries, which determine different characteristics of aroma or taste in commercial products. Biosynthesis of these compounds by alcoholysis...

  19. Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65 or Lecithin:Retinol Acyltransferase (LRAT.

    Directory of Open Access Journals (Sweden)

    Hendrik P N Scholl

    Full Text Available Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001. Safety and visual outcomes of a once-daily oral dose of 40 mg/m2/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44% showed a ≥20% increase and 4 of 18 (22% showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67% and 5 (28% of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI than non-responders (3.5 ± 1.2 μm. This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity.ClinicalTrials.gov NCT01014052.

  20. Alterations in high-density lipoprotein metabolism and reverse cholesterol transport in insulin resistance and type 2 diabetes mellitus : role of lipolytic enzymes, lecithin : cholesterol acyltransferase and lipid transfer proteins

    NARCIS (Netherlands)

    Borggreve, SE; de Vries, R; Dullaart, RPF

    2003-01-01

    Insulin resistance and type 2 diabetes mellitus are generally accompanied by low HDL cholesterol and high plasma triglycerides, which are major cardiovascular risk factors. This review describes abnormalities in HDL metabolism and reverse cholesterol transport, i.e. the transport of cholesterol from

  1. NCBI nr-aa BLAST: CBRC-TTRU-01-0581 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0581 ref|ZP_04756268.1| apolipoprotein N-acyltransferase [Francisella philomiragia subsp. philo...miragia ATCC 25015] ref|ZP_05249932.1| apolipoprotein N-acyltransferase [Francisella philo...miragia subsp. philomiragia ATCC 25015] gb|EET21657.1| apolipoprotein N-acyltransferase [Francisella philomiragia subsp. philomiragia ATCC 25015] ZP_04756268.1 0.014 24% ...

  2. Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Athenstaedt, Karin; Weys, Sabine; Paltauf, Fritz; Daum, Günther

    1999-01-01

    In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p presen...

  3. NCBI nr-aa BLAST: CBRC-MEUG-01-2833 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MEUG-01-2833 ref|YP_001931417.1| acyltransferase 3 [Sulfurihydrogenibium sp. Y...O3AOP1] gb|ACD66863.1| acyltransferase 3 [Sulfurihydrogenibium sp. YO3AOP1] YP_001931417.1 0.053 24% ...

  4. NCBI nr-aa BLAST: CBRC-MEUG-01-2127 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MEUG-01-2127 ref|YP_781289.1| acyltransferase 3 [Rhodopseudomonas palustris Bi...sA53] gb|ABJ06309.1| acyltransferase 3 [Rhodopseudomonas palustris BisA53] YP_781289.1 8.4 40% ...

  5. NCBI nr-aa BLAST: CBRC-CREM-01-1302 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1302 ref|YP_660676.1| acyltransferase 3 [Pseudoalteromonas atlantica T...6c] gb|ABG39622.1| acyltransferase 3 [Pseudoalteromonas atlantica T6c] YP_660676.1 3e-77 52% ...

  6. NCBI nr-aa BLAST: CBRC-OCUN-01-1157 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OCUN-01-1157 ref|ZP_01132590.1| acyltransferase, putative [Pseudoalteromonas t...unicata D2] gb|EAR29378.1| acyltransferase, putative [Pseudoalteromonas tunicata D2] ZP_01132590.1 1.1 32% ...

  7. NCBI nr-aa BLAST: CBRC-DNOV-01-0840 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0840 ref|YP_929162.1| acyltransferase family protein [Shewanella amazon...ensis SB2B] gb|ABM01493.1| acyltransferase family protein [Shewanella amazonensis SB2B] YP_929162.1 7.4 24% ...

  8. NCBI nr-aa BLAST: CBRC-CREM-01-1302 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1302 ref|YP_238152.1| Acyltransferase 3 [Pseudomonas syringae pv. syringa...e B728a] gb|AAY40114.1| Acyltransferase 3 [Pseudomonas syringae pv. syringae B728a] YP_238152.1 1e-75 48% ...

  9. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_001117159.1| acyltransferase 3 [Burkholderia vietnamiensis... G4] gb|ABO57694.1| acyltransferase 3 [Burkholderia vietnamiensis G4] YP_001117159.1 9e-64 54% ...

  10. NCBI nr-aa BLAST: CBRC-OPRI-01-0823 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OPRI-01-0823 ref|YP_002731074.1| apolipoprotein N-acyltransferase [Persephonel...la marina EX-H1] gb|ACO04611.1| apolipoprotein N-acyltransferase [Persephonella marina EX-H1] YP_002731074.1 0.48 26% ...

  11. NCBI nr-aa BLAST: CBRC-DNOV-01-0289 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0289 ref|YP_207241.1| putative lipo-oligosaccharide acyltransferase [Neisseria... gonorrhoeae FA 1090] gb|AAW88829.1| putative lipo-oligosaccharide acyltransferase [Neisseria gonorrhoeae FA 1090] YP_207241.1 4.5 21% ...

  12. NCBI nr-aa BLAST: CBRC-TTRU-01-0581 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0581 ref|YP_001676748.1| apolipoprotein N-acyltransferase [Francisella philomiragia subsp. phil...omiragia ATCC 25017] gb|ABZ86247.1| apolipoprotein N-acyltransferase [Francisella philomiragia subsp. philomiragia ATCC 25017] YP_001676748.1 0.011 25% ...

  13. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_001066820.1| acyltransferase family protein [Burkholderia ...pseudomallei 1106a] gb|ABN91330.1| acyltransferase family protein [Burkholderia pseudomallei 1106a] YP_001066820.1 1e-51 46% ...

  14. NCBI nr-aa BLAST: CBRC-XTRO-01-0669 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0669 ref|YP_741242.1| apolipoprotein N-acyltransferase [Alkalilimnicola... ehrlichei MLHE-1] gb|ABI55752.1| apolipoprotein N-acyltransferase [Alkalilimnicola ehrlichei MLHE-1] YP_741242.1 7e-05 33% ...

  15. NCBI nr-aa BLAST: CBRC-ETEL-01-1527 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ETEL-01-1527 ref|ZP_01733187.1| Putative acyltransferase [Flavobacteria bacter...ium BAL38] gb|EAZ96256.1| Putative acyltransferase [Flavobacteria bacterium BAL38] ZP_01733187.1 2.2 29% ...

  16. NCBI nr-aa BLAST: CBRC-PMAR-01-0774 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0774 ref|ZP_01733187.1| Putative acyltransferase [Flavobacteria bacter...ium BAL38] gb|EAZ96256.1| Putative acyltransferase [Flavobacteria bacterium BAL38] ZP_01733187.1 5.8 30% ...

  17. NCBI nr-aa BLAST: CBRC-TTRU-01-1149 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1149 ref|ZP_01733187.1| Putative acyltransferase [Flavobacteria bacter...ium BAL38] gb|EAZ96256.1| Putative acyltransferase [Flavobacteria bacterium BAL38] ZP_01733187.1 0.032 23% ...

  18. NCBI nr-aa BLAST: CBRC-CREM-01-1302 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1302 ref|YP_263931.1| possible acyltransferase family protein [Psychrobacter arctic...us 273-4] gb|AAZ18497.1| possible acyltransferase family protein [Psychrobacter arcticus 273-4] YP_263931.1 1e-80 52% ...

  19. NCBI nr-aa BLAST: CBRC-ACAR-01-1202 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-1202 ref|YP_001449434.1| putative acyltransferase [Streptococcus gordonii str. Challi...s substr. CH1] gb|ABV10561.1| putative acyltransferase [Streptococcus gordonii str. Challis substr. CH1] YP_001449434.1 1.9 25% ...

  20. NCBI nr-aa BLAST: CBRC-TTRU-01-0794 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0794 ref|YP_003092290.1| acyltransferase 3 [Pedobacter heparinus DSM 2...366] gb|ACU04228.1| acyltransferase 3 [Pedobacter heparinus DSM 2366] YP_003092290.1 0.11 24% ...

  1. AcEST: DK958057 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ot sp_hit_id Q42670 Definition sp|Q42670|PLSC_COCNU 1-acyl-sn-glycerol-3-phosphate acyltransferase OS=Cocos ...omo sapien... 31 3.9 >sp|Q42670|PLSC_COCNU 1-acyl-sn-glycerol-3-phosphate acyltransferase OS=Cocos nucifera

  2. NCBI nr-aa BLAST: CBRC-LAFR-01-0098 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0098 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 1.1 34% ...

  3. NCBI nr-aa BLAST: CBRC-LAFR-01-0247 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0247 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 2.5 34% ...

  4. NCBI nr-aa BLAST: CBRC-LAFR-01-1831 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1831 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 2.7 35% ...

  5. NCBI nr-aa BLAST: CBRC-MLUC-01-0475 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MLUC-01-0475 ref|ZP_01461366.1| 3-O-acyltransferase [Stigmatella aurantiaca DW...4/3-1] gb|EAU67913.1| 3-O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01461366.1 0.063 29% ...

  6. NCBI nr-aa BLAST: CBRC-LAFR-01-1192 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1192 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 2.3 35% ...

  7. NCBI nr-aa BLAST: CBRC-LAFR-01-1322 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1322 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 3.2 35% ...

  8. NCBI nr-aa BLAST: CBRC-LAFR-01-1422 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1422 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 0.82 36% ...

  9. NCBI nr-aa BLAST: CBRC-LAFR-01-1256 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1256 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 0.76 35% ...

  10. NCBI nr-aa BLAST: CBRC-LAFR-01-0561 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0561 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 2.3 36% ...

  11. NCBI nr-aa BLAST: CBRC-LAFR-01-0034 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0034 ref|ZP_01466199.1| mycarose O-acyltransferase [Stigmatella aurant...iaca DW4/3-1] gb|EAU63042.1| mycarose O-acyltransferase [Stigmatella aurantiaca DW4/3-1] ZP_01466199.1 1.2 35% ...

  12. NCBI nr-aa BLAST: CBRC-XTRO-01-0138 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|ZP_01001284.1| apolipoprotein N-acyltransferase [Oceanicola bats...Q01389.1| apolipoprotein N-acyltransferase [Oceanicola batsensis HTCC2597] gb|ABV95346.1| apolipoprotein N-a

  13. NCBI nr-aa BLAST: CBRC-XTRO-01-0530 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_984850.1| apolipoprotein N-acyltransferase [Acidovorax sp.... JS42] gb|ABM40774.1| apolipoprotein N-acyltransferase [Acidovorax sp. JS42] YP_984850.1 0.0 70% ...

  14. NCBI nr-aa BLAST: CBRC-XTRO-01-0530 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_972499.1| apolipoprotein N-acyltransferase [Acidovorax ave...nae subsp. citrulli AAC00-1] gb|ABM34725.1| apolipoprotein N-acyltransferase [Acidovorax avenae subsp. citrulli AAC00-1] YP_972499.1 1e-177 68% ...

  15. NCBI nr-aa BLAST: CBRC-XTRO-01-0530 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_550998.1| apolipoprotein N-acyltransferase [Polaromonas sp.... JS666] gb|ABE46100.1| apolipoprotein N-acyltransferase [Polaromonas sp. JS666] YP_550998.1 1e-147 60% ...

  16. NCBI nr-aa BLAST: CBRC-XTRO-01-0530 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_980683.1| apolipoprotein N-acyltransferase [Polaromonas na...phthalenivorans CJ2] gb|ABM35762.1| apolipoprotein N-acyltransferase [Polaromonas naphthalenivorans CJ2] YP_980683.1 1e-143 57% ...

  17. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_442494.1| acyltransferase family protein [Burkholderia thail...andensis E264] gb|ABC38658.1| acyltransferase family protein [Burkholderia thailandensis E264] YP_442494.1 2e-52 44% ...

  18. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_045598.1| putative acyltransferase [Acinetobacter sp. ADP1...] emb|CAG67776.1| putative acyltransferase [Acinetobacter sp. ADP1] YP_045598.1 1e-102 75% ...

  19. NCBI nr-aa BLAST: CBRC-CREM-01-1310 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1310 ref|YP_001086401.1| putative acyltransferase [Acinetobacter bauma...nnii ATCC 17978] gb|ABO13799.1| putative acyltransferase [Acinetobacter baumannii ATCC 17978] YP_001086401.1 1e-67 70% ...

  20. NCBI nr-aa BLAST: CBRC-CREM-01-1302 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1302 ref|NP_933100.1| acyltransferase family protein [Vibrio vulnificus... YJ016] dbj|BAC93071.1| acyltransferase family protein [Vibrio vulnificus YJ016] NP_933100.1 2e-74 48% ...

  1. NCBI nr-aa BLAST: CBRC-XTRO-01-0138 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|ZP_00961631.1| apolipoprotein N-acyltransferase [Roseovarius ...nubinhibens ISM] gb|EAP74993.1| apolipoprotein N-acyltransferase [Roseovarius nubinhibens ISM] ZP_00961631.1 1e-25 32% ...

  2. NCBI nr-aa BLAST: CBRC-XTRO-01-0138 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|ZP_01442058.1| apolipoprotein N-acyltransferase [Roseovarius ...sp. HTCC2601] gb|EAU47874.1| apolipoprotein N-acyltransferase [Roseovarius sp. HTCC2601] ZP_01442058.1 9e-31 31% ...

  3. NCBI nr-aa BLAST: CBRC-MDOM-08-0105 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-08-0105 ref|YP_002222709.1| apolipoprotein N-acyltransferase, putative [Borrelia recurrent...is A1] gb|ACH94488.1| apolipoprotein N-acyltransferase, putative [Borrelia recurrentis A1] YP_002222709.1 1.2 23% ...

  4. NCBI nr-aa BLAST: CBRC-XTRO-01-0530 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|ZP_01518111.1| apolipoprotein N-acyltransferase [Comamonas te...stosteroni KF-1] gb|EAV17580.1| apolipoprotein N-acyltransferase [Comamonas testosteroni KF-1] ZP_01518111.1 1e-139 55% ...

  5. Disease: H00158 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H00158 Lecithin:cholesterol acyltransferase deficiency; Norum disease; Fish-eye dise...ing to a triad of corneal dystrophy, hemolytic anemia, and proteinuria. Inherited metabolic dise

  6. Impaired suppression of plasma free fatty acids and triglycerides by acute hyperglycaemia-induced hyperinsulinaemia and alterations in high density lipoproteins in essential hypertension

    NARCIS (Netherlands)

    Ligtenberg, JJM; vanTol, A; vanHaeften, TW; Sluiter, WJ; Dullaart, RPF

    1996-01-01

    Objectives. Essential hypertension may be associated with abnormalities in free fatty acids (FFA) and triglyceride metabolism, which could lead to alterations in high density lipoproteins (HDL). Lecithin: cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are key factor

  7. Plasma phospholipid transfer protein activity is inversely associated with betaine in diabetic and non-diabetic subjects

    NARCIS (Netherlands)

    Dullaart, R. P. F.; Garcia, Erwin; Jeyarajah, Elias; Gruppen, Eke G.; Connelly, Margery A.

    2016-01-01

    Background: The choline metabolite, betaine, plays a role in lipid metabolism, and may predict the development of cardiovascular disease and type 2 diabetes mellitus (T2DM). Phospholipid transfer protein (PLTP) and lecithin: cholesterol acyltransferase (LCAT) require phosphatidylcholine as substrate

  8. Determination of a novel diacylglycerol acyltransferase 1 inhibitor, 2-[4-(4-{5-[2-phenyl-5-(trifluoromethyl) oxazole-4-carboxamido]-1H-benzo[d]imidazol-2-yl} phenyl) cyclohexyl] acetic acid (KR-69232) in rat plasma using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

    Science.gov (United States)

    Seo, Hyewon; Choi, Sung Heum; Kwak, Eun-Young; Zheng, Zhi; Kwak, Hyun Jung; Ahn, Jin Hee; Lee, Yong-Moon; Ahn, Sung-Hoon; Bae, Myung Ae; Song, Jin Sook

    2014-03-01

    A liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of KR-69232, a diacyltransferase 1 inhibitor, in rat plasma. KR-69232 in the concentration range of 0.004-4 µg/mL was linear. The intra-and inter-day precision and accuracy were acceptable (KR-69232 was stable under various storage and handling conditions. The method was applied successfully in a pharmacokinetic study of KR-69232 in rats.

  9. Studies on acyl-CoA: cholesterol acyltransferase inhibitors from microbial sourcesⅡ. Physico-chemical properties and structure elucidation of NA-209 compounds%微生物来源的胆固醇酰基转移酶抑制剂研究Ⅱ.NA-209A,B的理化特征和结构鉴别

    Institute of Scientific and Technical Information of China (English)

    唐勇; 杨大军; 姚天爵

    2000-01-01

    从曲霉H717的发酵产物中分离得到两个脂酰辅酶A:胆固醇酰基转移酶(ACAT)的抑制剂.根据EI-MS、FAB-MS和HREI-MS数据,确定其分子式分别为C27H33N3O7(分子量:511.2)和C27H33N3O5(分子量:479.2).综合紫外光谱、质谱、核磁共振光谱和X-结晶衍射等数据解析,确定其结构为含有吲哚环的二酮哌嗪类化合物.与文献对照,其中NA-209A为verruculogen的立体异构体,而NA-209B则为fumitremorgin B的立体异构体.

  10. Nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.

    Science.gov (United States)

    Nijland, Jeroen G; Ebbendorf, Bjorg; Woszczynska, Marta; Boer, Rémon; Bovenberg, Roel A L; Driessen, Arnold J M

    2010-11-01

    Industrial penicillin production levels by the filamentous fungus Penicillium chrysogenum increased dramatically by classical strain improvement. High-yielding strains contain multiple copies of the penicillin biosynthetic gene cluster that encodes three key enzymes of the β-lactam biosynthetic pathway. We have analyzed the gene cluster dose effect on penicillin production using the high-yielding P. chrysogenum strain DS17690 that was cured from its native clusters. The amount of penicillin V produced increased with the penicillin biosynthetic gene cluster number but was saturated at high copy numbers. Likewise, transcript levels of the biosynthetic genes pcbAB [δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase], pcbC (isopenicillin N synthase), and penDE (acyltransferase) correlated with the cluster copy number. Remarkably, the protein level of acyltransferase, which localizes to peroxisomes, was saturated already at low cluster copy numbers. At higher copy numbers, intracellular levels of isopenicillin N increased, suggesting that the acyltransferase reaction presents a limiting step at a high gene dose. Since the number and appearance of the peroxisomes did not change significantly with the gene cluster copy number, we conclude that the acyltransferase activity is limiting for penicillin biosynthesis at high biosynthetic gene cluster copy numbers. These results suggest that at a high penicillin production level, productivity is limited by the peroxisomal acyltransferase import activity and/or the availability of coenzyme A (CoA)-activated side chains.

  11. Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes

    DEFF Research Database (Denmark)

    Wei, Yongjun; Gossing, Michael; Bergenholm, David

    2017-01-01

    Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol(POS,C16:0C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18....... TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible...... for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast...

  12. DHHC protein-dependent palmitoylation protects regulator of G-protein signaling 4 from proteasome degradation

    OpenAIRE

    2010-01-01

    Regulator of G-protein signaling 4 (RGS4), an intracellular modulator of G-protein coupled receptor (GPCR)-mediated signaling, is regulated by multiple processes including palmitoylation and proteasome degradation. We found that co-expression of DHHC acyltransferases (DHHC3 or DHHC7), but not their acyltransferase-inactive mutants, increased expression levels of RGS4 but not its Cys2 to Ser mutant (RGS4C2S). DHHC3 interacts with and palmitoylates RGS4 but not RGS4C2S in vivo. Palmitoylation p...

  13. The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells

    Science.gov (United States)

    Raines, Sally A.; Hodgkinson, Michael R.; Dowle, Adam A.

    2017-01-01

    Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ’s acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family. PMID:28235057

  14. Understanding Acyl Chain and Glycerolipid Metabolism in Plants

    Energy Technology Data Exchange (ETDEWEB)

    Ohlrogge, John B.

    2013-11-05

    Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

  15. MsAcT in siliceous monolithic microreactors enables quantitative ester synthesis in water

    NARCIS (Netherlands)

    Szymańska, K.; Odrozek, K.; Zniszczoł, A.; Torrelo, G.; Resch, V.; Hanefeld, U.; Jarzębski, A.B.

    2016-01-01

    Acyltransferase from Mycobacterium smegmatis (MsAcT) immobilised in continuous-flow microchannel (30-50 μm dia.) reactors with hierarchical pore structure (4 cm3/g total pore volume) enabled quantitative, full and rapid transesterification of neopentylglycol with ethyl acetate in a biphasic 50/50 %

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

    Science.gov (United States)

    GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE (GPAT) genes encode enzymes involved in glycerolipid biosynthesis in plants. Ten GPAT homologues have been identified in Arabidopsis thaliana (Arabidopsis). GPATs 4-8 have been shown to be involved in the production of extracellular lipid barrier polyesters. Rece...

  17. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (Q921H8) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) THIKA_MOUSE 2e-57 ...

  18. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 1e-93 ...

  19. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (Q921H8) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) THIKA_MOUSE 2e-60 ...

  20. SwissProt search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 (P09110) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_HUMAN 1e-54 ...

  1. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 2e-51 ...

  2. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 2e-60 ...

  3. SwissProt search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 2e-56 ...

  4. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (Q8BWT1) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) THIM_MOUSE 2e-58 ...

  5. SwissProt search result: AK072460 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072460 J023121H13 (P13437) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) THIM_RAT 4e-55 ...

  6. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (P13437) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) THIM_RAT 7e-46 ...

  7. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 4e-94 ...

  8. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (Q05493) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YARLI 5e-51 ...

  9. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 1e-51 ...

  10. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (P21775) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) THIKA_RAT 1e-107 ...

  11. SwissProt search result: AK072460 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072460 J023121H13 (P42765) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) (T1) THIM_HUMAN 1e-57 ...

  12. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (P42765) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) (T1) THIM_HUMAN 7e-59 ...

  13. SwissProt search result: AK072460 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072460 J023121H13 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 2e-87 ...

  14. SwissProt search result: AK069768 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069768 J023030K20 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 6e-68 ...

  15. SwissProt search result: AK069768 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069768 J023030K20 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 2e-67 ...

  16. SwissProt search result: AK069768 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069768 J023030K20 (P13437) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) THIM_RAT 7e-48 ...

  17. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 2e-71 ...

  18. SwissProt search result: AK069768 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069768 J023030K20 (Q05493) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YARLI 6e-82 ...

  19. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 2e-51 ...

  20. SwissProt search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 (P21775) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) THIKA_RAT 1e-57 ...

  1. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 5e-72 ...

  2. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (Q570C8) 3-ketoacyl-CoA thiolase 5, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase 5) (Acetyl-CoA acyltransferase 5) (Peroxisomal 3-oxoacyl-CoA thiolase 5) THIK5_ARATH 1e-145 ...

  3. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (Q05493) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YARLI 5e-52 ...

  4. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (P13437) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) THIM_RAT 2e-62 ...

  5. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (Q05493) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YARLI 1e-115 ...

  6. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (P07871) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_RAT 3e-98 ...

  7. SwissProt search result: AK072460 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072460 J023121H13 (P07871) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_RAT 1e-113 ...

  8. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 1e-107 ...

  9. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (Q570C8) 3-ketoacyl-CoA thiolase 5, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase 5) (Acetyl-CoA acyltransferase 5) (Peroxisomal 3-oxoacyl-CoA thiolase 5) THIK5_ARATH 4e-85 ...

  10. SwissProt search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 3e-52 ...

  11. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (Q570C8) 3-ketoacyl-CoA thiolase 5, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se 5) (Acetyl-CoA acyltransferase 5) (Peroxisomal 3-oxoacyl-CoA thiolase 5) THIK5_ARATH 8e-52 ...

  12. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (P27796) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YEAST 4e-69 ...

  13. SwissProt search result: AK069768 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069768 J023030K20 (P42765) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) (T1) THIM_HUMAN 2e-49 ...

  14. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (Q8LF48) 3-ketoacyl-CoA thiolase 1, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase 1) (Acetyl-CoA acyltransferase 1) (Peroxisomal 3-oxoacyl-CoA thiolase 1) THIK1_ARATH 6e-87 ...

  15. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 1e-51 ...

  16. SwissProt search result: AK072460 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072460 J023121H13 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 6e-88 ...

  17. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (Q8LF48) 3-ketoacyl-CoA thiolase 1, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se 1) (Acetyl-CoA acyltransferase 1) (Peroxisomal 3-oxoacyl-CoA thiolase 1) THIK1_ARATH 3e-53 ...

  18. SwissProt search result: AK107395 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107395 002-127-D04 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 1e-86 ...

  19. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (P27796) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YEAST 4e-50 ...

  20. SwissProt search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 (Q8LF48) 3-ketoacyl-CoA thiolase 1, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase 1) (Acetyl-CoA acyltransferase 1) (Peroxisomal 3-oxoacyl-CoA thiolase 1) THIK1_ARATH 1e-157 ...

  1. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 3e-60 ...

  2. SwissProt search result: AK107395 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107395 002-127-D04 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 2e-86 ...

  3. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 3e-53 ...

  4. SwissProt search result: AK101017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK101017 J023149F12 (Q05493) 3-ketoacyl-CoA thiolase, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Peroxisomal 3-oxoacyl-CoA thiolase) THIK_YARLI 5e-51 ...

  5. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (P42765) 3-ketoacyl-CoA thiolase, mitochondrial (EC 2.3.1.16) (Beta-ketothiola...se) (Acetyl-CoA acyltransferase) (Mitochondrial 3-oxoacyl-CoA thiolase) (T1) THIM_HUMAN 5e-65 ...

  6. SwissProt search result: AK108135 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108135 002-139-E02 (Q8VCH0) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_MOUSE 3e-95 ...

  7. SwissProt search result: AK107395 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107395 002-127-D04 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 8e-87 ...

  8. SwissProt search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 (P33290) 3-ketoacyl-CoA thiolase A, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiol...ase A) (Acetyl-CoA acyltransferase A) (Peroxisomal 3-oxoacyl-CoA thiolase A) (Thiolase IA) THIKA_CANTR 4e-52 ...

  9. SwissProt search result: AK103194 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK103194 J033121P11 (P07871) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) THIKB_RAT 2e-60 ...

  10. SwissProt search result: AK102536 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK102536 J033096I21 (P33291) 3-ketoacyl-CoA thiolase B, peroxisomal precursor (EC 2.3.1.16) (Beta-ketothiola...se B) (Acetyl-CoA acyltransferase B) (Peroxisomal 3-oxoacyl-CoA thiolase B) (Thiolase IB) THIKB_CANTR 3e-53 ...

  11. The Cirque du Soleil of Golgi membrane dynamics.

    Science.gov (United States)

    Bankaitis, Vytas A

    2009-07-27

    The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid-specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.

  12. AcEST: BP915836 [AcEST

    Lifescience Database Archive (English)

    Full Text Available udouridine synthase OS=Leeuwenhoekiell... 33 5.8 tr|A8L3M4|A8L3M4_FRASN Acyltransferase 3 OS=Frankia sp. (st...9FLAO Pseudouridine synthase OS=Leeuwenhoekiella blandensis MED217 GN=MED217_04192 PE=3 SV=1 Length = 265 Sc

  13. Sizing up surfactant synthesis.

    Science.gov (United States)

    Han, SeungHye; Mallampalli, Rama K

    2014-08-01

    Phosphatidylcholine is generated through de novo synthesis and remodeling involving a lysophospholipid. In this issue of Cell Metabolism, research from the Shimizu lab (Harayama et al., 2014) demonstrates the highly selective enzymatic behavior of lysophospholipid acyltransferases. The authors present an enzymatic model for phosphatidylcholine molecular species diversification that impacts surfactant formation.

  14. Loss of NDG-4 extends lifespan and stress resistance in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Brejning, Jeanette; Nørgaard, Steffen; Schøler, Lone Vedel;

    2014-01-01

    NDG-4 is a predicted transmembrane acyltransferase protein that acts in the distribution of lipophilic factors. Consequently, ndg-4 mutants lay eggs with a pale appearance due to lack of yolk, and they are resistant to sterility caused by dietary supplementation with the long-chain omega-6 polyun...

  15. Overexpression of the tomato asc-1 gene mediates high insensitivity to AAL toxins and fumonisin B-1 in tomato hairy roots and confers resistance to Alternaria alternata f. sp lycopersici in Nicotiana umbratica plants

    NARCIS (Netherlands)

    Brandwagt, BF; Kneppers, TJA; Nijkamp, HJJ; Hille, J

    2002-01-01

    The sphinganine-analog mycotoxins (SAMs) fumonisin B-1 and AAL toxins are inhibitors of eukaryotic sphinganine N-acyltransferase in vitro. Treatment of eukaryotes with SAMs generally results in an accumulation of sphingoid base precursors and a depletion of complex sphingolipids. The asc,asc genotyp

  16. Overexpression of the Tomato Asc-1 Gene Mediates High Insensitivity to AAL Toxins and Fumonisin B1 in Tomato Hairy Roots and Confers Resistance to Alternaria alternata f. sp. lycopersici in Nicotiana umbratica Plants

    NARCIS (Netherlands)

    Brandwagt, Bas F.; Kneppers, Tarcies J.A.; Nijkamp, H. John J.; Hille, Jacques

    2002-01-01

    The sphinganine-analog mycotoxins (SAMs) fumonisin B1 and AAL toxins are inhibitors of eukaryotic sphinganine N-acyltransferase in vitro. Treatment of eukaryotes with SAMs generally results in an accumulation of sphingoid base precursors and a depletion of complex sphingolipids. The asc,asc genotype

  17. Moderate doses of alcoholic beverages with dinner and postprandial high density lipoprotein composition

    NARCIS (Netherlands)

    Hendriks, H.F.J.; Veenstra, J.; Tol, A. van; Groener, J.E.M.; Schaafsma, G.

    1998-01-01

    Moderate alcohol consumption is associated with a reduced risk of coronary heart disease. In this study, postprandial changes in plasma lipids, high-density lipoprotein (HDL) composition and cholesteryl ester transfer protein (CETP) and lecithin: cholesterol acyltransferase (LCAT) activity levels we

  18. NCBI nr-aa BLAST: CBRC-VPAC-01-0381 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available yltransferase [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] gb|ACQ67332.1| UDP-3-O-(3-hydroxym...yristoyl)-glucosamine N-acyltransferase [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] YP_002923480.1 0.76 28% ...

  19. NCBI nr-aa BLAST: CBRC-VPAC-01-0194 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available yltransferase [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] gb|ACQ67332.1| UDP-3-O-(3-hydroxym...yristoyl)-glucosamine N-acyltransferase [Candidatus Hamiltonella defensa 5AT (Acyrthosiphon pisum)] YP_002923480.1 0.58 29% ...

  20. Sequence Classification: 284440 [

    Lifescience Database Archive (English)

    Full Text Available A biosynthesis (KDO)2-(lauroyl)-lipid IVA acyltransferase || http://www.ncbi.nlm.nih.gov/protein/26248121 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|26248121|ref|NP_754161.1| Lipid

  1. Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome

    NARCIS (Netherlands)

    Lu, J.; Boeren, S.; Hooijdonk, van A.C.M.; Vervoort, J.J.M.; Hettinga, K.A.

    2015-01-01

    Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat con

  2. EST Table: AU000418 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available AU000418 e40523 11/12/09 GO hit GO:0016747(transferase activity, transferring acyl groups... other than amino-acyl groups) 10/09/28 100 %/162 aa emb|CBG34003.1| putative acyltransferase [Escheri

  3. A low-saturated-fat, low-cholesterol diet decreases plasma CETP activity and pre beta-HDL formation but does not affect cellular cholesterol efflux to plasma from type 1 diabetic patients

    NARCIS (Netherlands)

    De Vries, R; Beusekamp, BJ; Kerstens, MN; Groen, AK; Van Tol, A; Dullaart, RPF

    2005-01-01

    The aim of this study was to evaluate the effect of a low-saturated-fat, low-cholesterol diet on plasma lipopoproteins, pre beta-high density lipoprotein (HDL) formation, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP)

  4. Characterization of LpGPAT Gene in Lilium pensylvanicum and Response to Cold Stress

    Directory of Open Access Journals (Sweden)

    Shao-kun Sun

    2015-01-01

    Full Text Available LpGPAT was obtained from L. pensylvanicum using RT-PCR and rapid amplification of cDNA ends. The cloned full-length cDNA was 1544 bp; it encoded 410 amino acids and had a molecular size of 46 KDa. The nucleic acid sequence analysis showed that it shared high homology with other known GPATs. SMAT result suggests that there is a PlsC that exists in 176-322 amino acid sequence of LpGAPT; it means LpGPAT protein is a member of the family of acyltransferase and has acyltransferase enzymatic activity. Result of real-time quantitative PCR and semiquantitative PCR support LpGPAT gene is definitely induced by low temperature stress.

  5. Studies of association of AGPAT6 variants with type 2 diabetes and related metabolic phenotypes in 12,068 Danes

    DEFF Research Database (Denmark)

    Snogdal, Lena Sønder; Grarup, Niels; Banasik, Karina;

    2013-01-01

    Type 2 diabetes, obesity and insulin resistance are characterized by hypertriglyceridemia and ectopic accumulation of lipids in liver and skeletal muscle. AGPAT6 encodes a novel glycerol-3 phosphate acyltransferase, GPAT4, which catalyzes the first step in the de novo triglyceride synthesis. AGPA......-deficient mice show lower weight and resistance to diet- and genetically induced obesity. Here, we examined whether common or low-frequency variants in AGPAT6 associate with type 2 diabetes or related metabolic traits in a Danish population.......Type 2 diabetes, obesity and insulin resistance are characterized by hypertriglyceridemia and ectopic accumulation of lipids in liver and skeletal muscle. AGPAT6 encodes a novel glycerol-3 phosphate acyltransferase, GPAT4, which catalyzes the first step in the de novo triglyceride synthesis. AGPAT6...

  6. Altered retinol status and expression of retinol-related proteins in streptozotocin-induced type 1 diabetic model rats

    OpenAIRE

    Takitani, Kimitaka; Inoue, Keisuke; Koh, Maki; Miyazaki, Hiroshi; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2015-01-01

    Diabetes is a metabolic disorder characterized by chronic hyperglycemia. Advanced diabetes is associated with severe complications and impaired nutritional status. Here, we assessed the expression of retinol-associated proteins, including β-carotene 15,15'-monooxygenase (BCMO), lecithin:retinol acyltransferase (LRAT), aldehyde dehydrogenase (ALDH), and cytochrome P450 26A1 (CYP26A1), and measured retinol levels in the plasma and liver of streptozotocin (STZ)-induced type 1 diabetic model rats...

  7. Targeting Mechanisms of Resistance to Taxane-Based Chemotherapy

    Science.gov (United States)

    2006-09-01

    function to eliminate small organic cations and an arrays of toxic compounds . Glycine-N-acyltransferase (GLYAT) modulates the conjugation of glycine to...member A (torsin A) 1.19 0.00066 Hs.444600 LAT1-3TM LAT1-3TM protein 1.18 0.00055 Hs.517948 DHX30 DEAH (Asp-Glu-Ala-His) box polypeptide 30 1.18

  8. Genome Annotation and Transcriptomics of Oil-Producing Algae

    Science.gov (United States)

    2015-03-16

    Collaborators Christoph Benning (Michigan State University) for chemical analysis of fatty acids in TAG and TAG quantitation Arthur Grossman (Carnegie... Grossman , A.R., Merchant, S.S. (2012) Three acyltransferases and a nitrogen responsive regulator are implicated in nitrogen starvation-induced...Hong- Hermesdorf, A., Shaw, J., Karpowicz, S.J., Gallaher, S.D., Johnson, S., Benning, C., Pellegrini, M., Grossman , A.R., Merchant, S.S. (2012

  9. Targeted Approaches to Overcoming Endocrine Resistance in Breast Cancer

    Science.gov (United States)

    2012-08-01

    to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data...needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this...Diacylglycerol O-acyltransferase homolog 1 XM_001719374 EXOSC4 Exosome component 4 BM911415 FAM82B Family with sequence similarity 82, member B

  10. KirCII- promising tool for polyketide diversification

    DEFF Research Database (Denmark)

    Musiol-Kroll, Ewa Maria; Härtner, Thomas; Kulik, Andreas;

    2014-01-01

    Kirromycin is produced by Streptomyces collinus Tü 365. This compound is synthesized by a large assembly line of type I polyketide synthases and non-ribosomal peptide synthetases (PKS I/NRPS), encoded by the genes kirAI-kirAVI and kirB. The PKSs KirAI-KirAV have no acyltransferase domains integra...... introducing the non-native substrates in an in vivo context. Thus, KirCII represents a promising tool for polyketide diversification....

  11. Triterpenic Acids Present in Hawthorn Lower Plasma Cholesterol by Inhibiting Intestinal ACAT Activity in Hamsters

    OpenAIRE

    Yuguang Lin; Vermeer, Mario A.; Trautwein, Elke A.

    2011-01-01

    Hawthorn (Crataegus pinnatifida) is an edible fruit used in traditional Chinese medicine to lower plasma lipids. This study explored lipid-lowering compounds and underlying mechanisms of action of hawthorn. Hawthorn powder extracts inhibited acylCoA:cholesterol acyltransferase (ACAT) activity in Caco-2 cells. The inhibitory activity was positively associated with triterpenic acid (i.e., oleanolic acid (OA) and ursolic acid (UA)) contents in the extracts. Cholesterol lowering effects of hawtho...

  12. Biological properties of lipoic acid

    Directory of Open Access Journals (Sweden)

    Anna Bilska

    2002-06-01

    Full Text Available Lipoic acid is a prostetic group of H-protein of the glycine cleavage system and the dihydrolipoamide acyltransferases (E2 of the pyruvate, alpha-ketoglutarate and branched-chain alpha-keto acid dehydrogenase complexes. Lipoic acid and its reduced form, dihydrolipoic acid, reacts with oxygen reactive species. This paper reviews the beneficial effects in oxidative stress models or clinical conditions.

  13. Isolation and Identification of Prenylflavonoids from Humulus lupulus

    Institute of Scientific and Technical Information of China (English)

    WANG Wen-Shu; ZHOU Ya-Wei; YE Yun-Hua; LI Mei-Lan

    2003-01-01

    @@ The hop plant (Humulus lupulus L. Cannabinaceae ) is cultivated widely through the temperate zones of the world. The female inflorescences of the hop plant (hops) are used in the brewing industry to give bitterness and aroma of beer. In China, hops are used as stomachics, diuretic and tranquilizer. [1] A number of prenylflavonoids have been isolated from hops, [2,3] which caused attention because of their potential anti-cancer properties, [4] endocrine activities, [5] and diacylglycerol acyltransferase inhibition. [6

  14. Host cells and methods for producing isoprenyl alkanoates

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

    2015-12-01

    The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

  15. The identification of cutin synthase: formation of the plant polyester cutin

    DEFF Research Database (Denmark)

    Yeats, Trevor H.; Martin, Laetitia B. B.; Viart, Helene Marie-France;

    2012-01-01

    A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular...... acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol. CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase....

  16. The identification of cutin synthase: formation of the plant polyester cutin.

    Science.gov (United States)

    Yeats, Trevor H; Martin, Laetitia B B; Viart, Hélène M-F; Isaacson, Tal; He, Yonghua; Zhao, Lingxia; Matas, Antonio J; Buda, Gregory J; Domozych, David S; Clausen, Mads H; Rose, Jocelyn K C

    2012-07-01

    A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol. CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase.

  17. The identification of cutin synthase: formation of the plant polyester cutin

    OpenAIRE

    Yeats, Trevor H.; Martin, Laetitia B. B.; Viart, Hélène M.-F.; Isaacson, Tal; He, Yonghua; Zhao, Lingxia; Matas, Antonio J.; Buda, Gregory J.; Domozych, David S.; Clausen, Mads H.; Rose, Jocelyn K. C.

    2012-01-01

    A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol (2-MHG). CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating th...

  18. Intrinsic Cholinergic Mechanisms Regulating Cerebral Blood Flow as a Target for Organo Phosphate Action.

    Science.gov (United States)

    1986-10-01

    Harvard Apparatus, Model 680), the animals were paralyzed with tubocurarine (0.5 mg/kg, i.m., initially; supplemented with 0.2 mg/kg hourly), and...another acetyltransferase, carnitine acetyltransferase (EC. 2.3.1.7) (Bresolin et al., 1982). -17- In some experiments the selective inhibitor of ChAT, 4... Carnitine , carnitine acyltransferases, and rat brain function. Exp. Neurol. 78, 285-292. Briggs, C.A. and Cooper, J.A. (1981) A synaptosomal preparation

  19. Influence of Nutritional Factors on Lipid Metabolism.

    Science.gov (United States)

    1980-12-01

    conditions of chronic high level fat oxidation such as exercise, Askew et al. (121) fed exercising rats diets supplemented with 0.5Z L- carnitine . Although...exercise increased adipose tissue fatty acid turnover, supplemental dietary carnitine neither increased skeletal muscle in vitro fatty acid oxidation...some investigators believe the relative activities of the sn-glycerolphosphate acyltransferase and carnitine palmttyltrans- ferase may influence the

  20. Altered Lipid and Salt Taste Responsivity in Ghrelin and GOAT Null Mice

    OpenAIRE

    Huan Cai; Wei-Na Cong; Caitlin M Daimon; Rui Wang; Tschöp, Matthias H.; Jean Sévigny; Bronwen Martin; Stuart Maudsley

    2013-01-01

    Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both...

  1. Polymorphisms in lipogenic genes and milk fatty acid composition in Holstein dairy cattle.

    Science.gov (United States)

    Nafikov, Rafael A; Schoonmaker, Jon P; Korn, Kathleen T; Noack, Kristin; Garrick, Dorian J; Koehler, Kenneth J; Minick-Bormann, Jennifer; Reecy, James M; Spurlock, Diane E; Beitz, Donald C

    2014-12-01

    Changing bovine milk fatty acid (FA) composition through selection can decrease saturated FA (SFA) consumption, improve human health and provide a means for manipulating processing properties of milk. Our study determined associations between milk FA composition and genes from triacylglycerol (TAG) biosynthesis pathway. The GC dinucleotide allele of diacylglycerol O-acyltransferase 1:g.10433-10434AA >GC was associated with lower palmitic acid (16:0) concentration but higher oleic (18:1 cis-9), linoleic (18:2 cis-9, cis-12) acid concentrations, and elongation index. Accordingly, the GC dinucleotide allele was associated with lower milk fat percentage and SFA concentrations but higher monounsaturated FA and polyunsaturated FA (PUFA) concentrations. The glycerol-3-phosphate acyltransferase, mitochondrial haplotypes were associated with higher myristoleic acid (14:1 cis-9) concentration and C14 desaturation index. The 1-acylglycerol-3-phosphate acyltransferase 1 haplotypes were associated with higher PUFA and linoleic acid concentrations. The results of this study provide information for developing genetic tools to modify milk FA composition in dairy cattle.

  2. Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway.

    Science.gov (United States)

    Cagliari, Alexandro; Margis-Pinheiro, Márcia; Loss, Guilherme; Mastroberti, Alexandra Antunes; de Araujo Mariath, Jorge Ernesto; Margis, Rogério

    2010-11-01

    Castor bean (Ricinus communis) oil contains ricinoleic acid-rich triacylglycerols (TAGs). As a result of its physical and chemical properties, castor oil and its derivatives are used for numerous bio-based products. In this study, we survey the Castor Bean Genome Database to report the identification of TAG biosynthesis genes. A set of 26 genes encoding six distinct classes of enzymes involved in TAGs biosynthesis were identified. In silico characterization and sequence analysis allowed the identification of plastidic isoforms of glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase enzyme families, involved in the prokaryotic lipid biosynthesis pathway, that form a cluster apart from the cytoplasmic isoforms, involved in the eukaryotic pathway. In addition, two distinct membrane bound diacylglycerol acyltransferase enzymes were identified. Quantitative expression pattern analyses demonstrated variations in gene expressions during castor seed development. A tendency of maximum expression level at the middle of seed development was observed. Our results represent snapshots of global transcriptional activities of genes encompassing six enzyme families involved in castor bean TAG biosynthesis that are present during seed development. These genes represent potential targets for biotechnological approaches to produce nutritionally and industrially desirable oils.

  3. Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

    Energy Technology Data Exchange (ETDEWEB)

    Byers, D.M.; Meighen, E.A.

    1985-09-01

    Pulse-chase experiments with (/sup 3/H)tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a /sup 3/H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi (/sup 3/H)acylprotein and (/sup 3/H)palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (34K) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.

  4. The HRASLS (PLA/AT) subfamily of enzymes.

    Science.gov (United States)

    Mardian, Emily B; Bradley, Ryan M; Duncan, Robin E

    2015-10-26

    The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1-5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A1/2 (PLA1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.

  5. Cloning and Characterization of Porcine TSARG7 Gene and Analysis of Its Tissue-Specific Expression

    Institute of Scientific and Technical Information of China (English)

    LI Mei-li; LI Gui-qiang; FANG Wei; WANG Wei; SONG Xiao-guang; LI Er-lin; JIA Chao; XU Yin-xue

    2009-01-01

    TSARG7 is a novel member of the acyltransferase family since its sequence possesses the highly conserved phosphate acyltransferase (PIsC) domain existing in all acyltransferase-like proteins. The porcine TSARG7 had been identified by cloning in silico but had not been confirmed experimentally. The full-length mRNA of porcine TSARG7 gene was sequenced and two splice variants were discovered. The full-length cDNA of TSARG7 variant 1 was 2 513 bp and variant 2 was 2 634 bp. The putative porcine TSARG7 proteins, which were located in the cytoplasm, encoded 458 and 456 amino acids, respectively. Real-time PCR analysis showed that TSARG7 gene was expressed in various tissues, but at different levels. The expression levels of this gene were higher in the skeletal muscle, heart, and testis than that in other tissues, suggesting that the TSARG7 gene played a role in procine skeletal muscle, heart, and testis functions.

  6. Detailed characterization of the substrate specificity of mouse wax synthase.

    Science.gov (United States)

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  7. Novel eukaryotic enzymes modifying cell-surface biopolymers

    Directory of Open Access Journals (Sweden)

    Aravind L

    2010-01-01

    Full Text Available Abstract Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA. We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1 the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58, which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber

  8. Novel eukaryotic enzymes modifying cell-surface biopolymers

    Science.gov (United States)

    2010-01-01

    Background Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins. Results Using sequence analysis and conservation we define the acyltransferase domain prototyped by the fungal Cas1p proteins, identify its active site residues and unify them to the superfamily of classical 10TM acyltransferases (e.g. oatA). We also identify a novel family of esterases (prototyped by the previously uncharacterized N-terminal domain of Cas1p) that have a similar fold as the SGNH/GDSL esterases but differ from them in their conservation pattern. Conclusions We posit that the combined action of the acyltransferase and esterase domain plays an important role in controlling the acylation levels of glycans and thereby regulates their physico-chemical properties such as hygroscopicity, resistance to enzymatic hydrolysis and physical strength. We present evidence that the action of these novel enzymes on glycans might play an important role in host-pathogen interaction of plants, fungi and metazoans. We present evidence that in plants (e.g. PMR5 and ESK1) the regulation of carbohydrate acylation by these acylesterases might also play an important role in regulation of transpiration and stress resistance. We also identify a subfamily of these esterases in metazoans (e.g. C7orf58), which are fused to an ATP-grasp amino acid ligase domain that is predicted to catalyze, in certain animals, modification of cell surface polymers by amino acid or peptides. Reviewers This article was reviewed by Gaspar Jekely and Frank Eisenhaber PMID:20056006

  9. Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice.

    Science.gov (United States)

    Harada, Kenji; Shen, Wen-Jun; Patel, Shailja; Natu, Vanita; Wang, Jining; Osuga, Jun-ichi; Ishibashi, Shun; Kraemer, Fredric B

    2003-12-01

    To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSL-deficient (HSL-/-) and wild-type mice were fed normal chow or high-fat diets. HSL-/- mice were resistant to diet-induced obesity showing higher core body temperatures. Weight and triacylglycerol contents were decreased in white adipose tissue (WAT) but increased in both brown adipose tissue (BAT) and liver of HSL-/- mice. Serum insulin levels in the fed state and tumor necrosis factor-alpha mRNA levels in adipose tissues were higher, whereas serum levels of adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin and leptin, as well as mRNA levels of ACRP30/adiponectin, leptin, resistin, and adipsin in WAT, were lower in HSL-/- mice than in controls. Expression of transcription factors associated with adipogenesis (peroxisome proliferator-activated receptor-gamma, CAAT/enhancer-binding protein-alpha) and lipogenesis (carbohydrate response element-binding protein, adipocyte determination- and differentiation-dependent factor-1/sterol regulatory element-binding protein-1c), as well as of adipose differentiation markers (adipocyte lipid-binding protein, perilipin, lipoprotein lipase), lipogenic enzymes (glycerol-3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase-1 and -2, fatty acid synthase, ATP citrate lyase) and insulin signaling proteins (insulin receptor, insulin receptor substrate-1, GLUT4), was suppressed in WAT but not in BAT of HSL-/- mice. In contrast, expression of genes associated with cholesterol metabolism (sterol-regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase-1) and thermogenesis (uncoupling protein-2) was upregulated in both WAT and BAT of HSL-/- mice. Our results suggest that impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels.

  10. Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes.

    Science.gov (United States)

    Wei, Yongjun; Gossing, Michael; Bergenholm, David; Siewers, Verena; Nielsen, Jens

    2017-12-01

    Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.

  11. Human trifunctional protein alpha links cardiolipin remodeling to beta-oxidation.

    Directory of Open Access Journals (Sweden)

    William A Taylor

    Full Text Available Cardiolipin (CL is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL to CL and was identical to the alpha subunit of trifunctional protein (αTFP lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.

  12. Substrate recognition by the cell surface palmitoyl transferase DHHC5.

    Science.gov (United States)

    Howie, Jacqueline; Reilly, Louise; Fraser, Niall J; Vlachaki Walker, Julia M; Wypijewski, Krzysztof J; Ashford, Michael L J; Calaghan, Sarah C; McClafferty, Heather; Tian, Lijun; Shipston, Michael J; Boguslavskyi, Andrii; Shattock, Michael J; Fuller, William

    2014-12-09

    The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼ 120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme-substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump.

  13. Using the candidate gene approach for detecting genes underlying seed oil concentration and yield in soybean.

    Science.gov (United States)

    Eskandari, Mehrzad; Cober, Elroy R; Rajcan, Istvan

    2013-07-01

    Increasing the oil concentration in soybean seeds has been given more attention in recent years because of demand for both edible oil and biodiesel production. Oil concentration in soybean is a complex quantitative trait regulated by many genes as well as environmental conditions. To identify genes governing seed oil concentration in soybean, 16 putative candidate genes of three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl-CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) involved in triacylglycerol (TAG) biosynthesis pathways were selected and their sequences retrieved from the soybean database ( http://www.phytozome.net/soybean ). Three sequence mutations were discovered in either coding or noncoding regions of three DGAT soybean isoforms when comparing the parents of a 203 recombinant inbreed line (RIL) population; OAC Wallace and OAC Glencoe. The RIL population was used to study the effects of these mutations on seed oil concentration and other important agronomic and seed composition traits, including seed yield and protein concentration across three field locations in Ontario, Canada, in 2009 and 2010. An insertion/deletion (indel) mutation in the GmDGAT2B gene in OAC Wallace was significantly associated with reduced seed oil concentration across three environments and reduced seed yield at Woodstock in 2010. A mutation in the 3' untranslated (3'UTR) region of GmDGAT2C was associated with seed yield at Woodstock in 2009. A mutation in the intronic region of GmDGAR1B was associated with seed yield and protein concentration at Ottawa in 2010. The genes identified in this study had minor effects on either seed yield or oil concentration, which was in agreement with the quantitative nature of the traits. However, the novel gene-specific markers designed in the present study can be used in soybean breeding for marker-assisted selection aimed at increasing seed yield and oil

  14. Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumour brain tissue

    DEFF Research Database (Denmark)

    Petersen, G.; Moesgaard, B.; Hansen, Harald S.

    2005-01-01

    The endogenous levels of the two cannabinoid receptor ligands 2-arachidonoyl glycerol and anandamide, and their respective congeners, monoacyl glycerols and N-acylethanolamines, as well as the phospholipid precursors of N-acylethanolamines, were measured by gas chromatography-mass spectrometry in...... in glioblastoma (WHO grade IV) tissue and meningioma (WHO grade I) tissue and compared with human non-tumour brain tissue. Furthermore, the metabolic turnover of N-acylethanolamines was compared by measurements of the enzymatic activity of N-acyltransferase, N...

  15. Cloning of Tung Tree DGAT2 Gene and Construction of RNAi Binary Expression Vector with Convergent Promoters%油桐DGAT2基因克隆及其RNAi双元表达载体构建

    Institute of Scientific and Technical Information of China (English)

    徐玲娜; 汪阳东; 陈益存; 张姗姗

    2011-01-01

    To identify the physiological function of Diacylglycerol Acyltransferase 2 (DGAT2) coding gene in the process of tung oil biosynthesis, DGAT2 was cloned from cDNA of tung tree kernel and then linked with pMD18-T vector for sequen-cing. The 969bp fragment containing Open Reading Frame was acquired. Subsequently, RNAi binary expression vector pD35-DGAT2 was constructed, which expressed DGAT2 in two opposite ways. The studies provide the possibilities to fur-ther identify the function of DGAT2 in tung oil biosynthesis by RNAi technology and hold promise for genetic engineering of Venicia fordii.

  16. PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast

    DEFF Research Database (Denmark)

    Le Guédard, Marina; Bessoule, Jean-Jacques; Boyer, Valérie;

    2009-01-01

    In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either...... phosphatidylinositol is synthesized from distinct cytidine diphosphate-diacylglycerol molecules, or that, after its synthesis, it is modified by a hypothetical acyltransferase that incorporates saturated fatty acid into neo-synthesized molecules of phosphatidylinositol. We used database search methods to identify...... as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol....

  17. Reference: 782 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 782 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u4ria224u18621978i Li Fenglin...ulate to high concentrations in the seed oils of a few plant species, including j...e report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransfe... of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Sacch...ntification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransfe

  18. CtBP/BARS: a dual-function protein involved in transcription co-repression and Golgi membrane fission

    OpenAIRE

    Nardini, Marco; Spanò, Stefania; Cericola, Claudia; Pesce, Alessandra; Massaro, Anna; Millo, Enrico; Luini, Alberto; Corda, Daniela; Bolognesi, Martino

    2003-01-01

    C-terminal-binding protein/brefeldin A-ADP ribosylated substrate (CtBP/BARS) plays key roles in development and oncogenesis as a transcription co-repressor, and in intracellular traffic as a promoter of Golgi membrane fission. Co-repressor activity is regulated by NAD(H) binding to CtBP/BARS, while membrane fission is associated with its acyl-CoA-dependent acyltransferase activity. Here, we report the crystal structures of rat CtBP/BARS in a binary complex with NAD(H), and in a ternary comple...

  19. Brain levels of N-acylethanolamine phospholipids in mice during pentylenetetrazol-induced seizure

    DEFF Research Database (Denmark)

    Moesgaard, B.; Hansen, H.H.; Petersen, G.

    2003-01-01

    The N-acylethanolamine phospholipids (NAPE) are precursors for N-acylethanolamines (NAE), including anandamide (20:4-NAE), which is a ligand for the cannabinoid receptors. Previously, NAPE were believed to be found only in injured tissue, e.g., after neurodegenerative insults. Neuronal injury may...... and of N-acyltransferase in brain membrane preparations from adult and 3-d-old mice revealed an enzyme pattern in the adult mice that was favorable for NAE accumulation as opposed to NAPE accumulation. Thus, there was no difference in NAPE levels; at present, however, this does not exclude that NAE may...

  20. AcEST: BP912229 [AcEST

    Lifescience Database Archive (English)

    Full Text Available n tr|Q94589|Q94589_9CILI Lembadion factor OS=Lembadion bullinum Align length 41 S......................................done Score E Sequences producing significant alignments: (bits) Value tr|Q94589|Q94589_9CILI Lemba...dion factor OS=Lembadion bullinum GN... 34 4.8 tr|B2IJB4...|B2IJB4_BEII9 Acyltransferase 3 OS=Beijerinckia indica ... 33 8.2 >tr|Q94589|Q94589_9CILI Lembadion factor OS=Lemba

  1. American Chemical Society--238th National Meeting & Exposition. Developments in medicinal chemistry: part 2. 16-20 August 2009, Washington DC, USA.

    Science.gov (United States)

    Gater, Deborah; Macauley, Donald

    2009-10-01

    The 238th National Meeting and Exposition of the American Chemical Society, held in Washington DC, included topics covering new compounds and developments in the field of medicinal chemistry. This conference report highlights selected presentations on inhibitors of PARP, a heme oxygenase 1 (HO-1) inhibitor, NS3 protease inhibitors, a corticotrophin-releasing factor 1 (CRF-1) receptor antagonist, a cannabinoid receptor antagonist, diacylglycerol acyltransferase inhibitors, cathepsin and chymase receptor inhibitors, and MAPK inhibitors. Investigational drugs discussed include veliparib (Abbott Laboratories), MK-4827 (Merck & Co Inc), OB-24 (Osta Biotechnologies), BMS-339, BMS-764459, BMS-812204 and BMS-640994 (all Bristol-Myers Squibb Co), and JNJ-10311795 (Johnson & Johnson).

  2. Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis.

    Science.gov (United States)

    Lippold, Felix; vom Dorp, Katharina; Abraham, Marion; Hölzl, Georg; Wewer, Vera; Yilmaz, Jenny Lindberg; Lager, Ida; Montandon, Cyrille; Besagni, Céline; Kessler, Felix; Stymne, Sten; Dörmann, Peter

    2012-05-01

    During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.

  3. Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes.

    Science.gov (United States)

    Shinohara, Naoki; Uyama, Toru; Jin, Xing-Hua; Tsuboi, Kazuhito; Tonai, Takeharu; Houchi, Hitoshi; Ueda, Natsuo

    2011-11-01

    A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

  4. Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

    Science.gov (United States)

    Imamura, Sousuke; Kawase, Yasuko; Kobayashi, Ikki; Sone, Toshiyuki; Era, Atsuko; Miyagishima, Shin-Ya; Shimojima, Mie; Ohta, Hiroyuki; Tanaka, Kan

    2015-10-01

    Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae.

  5. A polyketide synthase-peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles.

    Science.gov (United States)

    Piel, Jörn

    2002-10-29

    Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. The antitumor polyketides of the pederin family, isolated from beetles and sponges, are an example. Drug development from such sources is commonly hampered by low yields and the difficulty of sustaining invertebrate cultures. To obtain insight into the true producer and find alternative supplies of these rare drug candidates, the putative pederin biosynthesis genes were cloned from total DNA of Paederus fuscipes beetles, which use this compound for chemical defense. Sequence analysis of the gene cluster and adjacent regions revealed the presence of ORFs with typical bacterial architecture and homologies. The ped cluster, which is present only in beetle specimens with high pederin content, is located on a 54-kb region bordered by transposase pseudogenes and encodes a mixed modular polyketide synthase/nonribosomal peptide synthetase. Notably, none of the modules contains regions with homology to acyltransferase domains, but two copies of isolated monodomain acyltransferase genes were found at the upstream end of the cluster. In line with an involvement in pederin biosynthesis, the upstream cluster region perfectly mirrors pederin structure. The unexpected presence of additional polyketide synthase/nonribosomal peptide synthetase modules reveals surprising insights into the evolutionary relationship between pederin-type pathways in beetles and sponges.

  6. Controlled buckling structures in semiconductor interconnects and nanomembranes for stretchable electronics

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, John A.; Meitl, Matthew; Sun, Yugang; Ko, Heung Cho; Carlson, Andrew; Choi, Won Mook; Stoykovich, Mark; Jiang, Hanqing; Huang, Yonggang; Nuzzo, Ralph G.; Zhu, Zhengtao; Menard, Etienne; Khang, Dahl-Young

    2016-04-26

    The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

  7. Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao,Y.; Jogl, G.; Tong, L.

    2006-01-01

    Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

  8. DGAT2 Mutation in a Family with Autosomal-Dominant Early-Onset Axonal Charcot-Marie-Tooth Disease.

    Science.gov (United States)

    Hong, Young Bin; Kang, Junghee; Kim, Ji Hyun; Lee, Jinho; Kwak, Geon; Hyun, Young Se; Nam, Soo Hyun; Hong, Hyun Dae; Choi, Yu-Ri; Jung, Sung-Chul; Koo, Heasoo; Lee, Ji Eun; Choi, Byung-Ok; Chung, Ki Wha

    2016-05-01

    Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy and is a genetically and clinically heterogeneous disorder. We examined a Korean family in which two individuals had an autosomal-dominant axonal CMT with early-onset, sensory ataxia, tremor, and slow disease progression. Pedigree analysis and exome sequencing identified a de novo missense mutation (p.Y223H) in the diacylglycerol O-acyltransferase 2 (DGAT2) gene. DGAT2 encodes an endoplasmic reticulum-mitochondrial-associated membrane protein, acyl-CoA:diacylglycerol acyltransferase, which catalyzes the final step of the triglyceride (TG) biosynthesis pathway. The patient showed consistently decreased serum TG levels, and overexpression of the mutant DGAT2 significantly inhibited the proliferation of mouse motor neuron cells. Moreover, the variant form of human DGAT2 inhibited the axonal branching in the peripheral nervous system of zebrafish. We suggest that mutation of DGAT2 is the novel underlying cause of an autosomal-dominant axonal CMT2 neuropathy. This study will help provide a better understanding of the pathophysiology of axonal CMT and contribute to the molecular diagnostics of peripheral neuropathies.

  9. Brain Natriuretic Peptide Stimulates Lipid Metabolism through Its Receptor NPR1 and the Glycerolipid Metabolism Pathway in Chicken Adipocytes.

    Science.gov (United States)

    Huang, H Y; Zhao, G P; Liu, R R; Li, Q H; Zheng, M Q; Li, S F; Liang, Z; Zhao, Z H; Wen, J

    2015-11-03

    Brain natriuretic peptide (BNP) is related to lipid metabolism in mammals, but its effect and the molecular mechanisms underlying it in chickens are incompletely understood. We found that the level of natriuretic peptide precursor B (NPPB, which encodes BNP) mRNA expression in high-abdominal-fat chicken groups was significantly higher than that of low-abdominal-fat groups. Partial correlations indicated that changes in the weight of abdominal fat were positively correlated with NPPB mRNA expression level. In vitro, compared with the control group, preadipocytes with NPPB interference showed reduced levels of proliferation, differentiation, and glycerin in media. Treatments of cells with BNP led to enhanced proliferation and differentiation of cells and glycerin concentration, and mRNA expression of its receptor natriuretic peptide receptor 1 (NPR1) was upregulated significantly. In cells exposed to BNP, 482 differentially expressed genes were identified compared with controls without BNP. Four genes known to be related to lipid metabolism (diacylglycerol kinase; lipase, endothelial; 1-acylglycerol-3-phosphate O-acyltransferase 1; and 1-acylglycerol-3-phosphate O-acyltransferase 2) were enriched in the glycerolipid metabolism pathway and expressed differentially. In conclusion, BNP stimulates the proliferation, differentiation, and lipolysis of preadipocytes through upregulation of the levels of expression of its receptor NPR1 and key genes enriched in the glycerolipid metabolic pathway.

  10. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway

    Directory of Open Access Journals (Sweden)

    Bourgaud Frédéric

    2009-03-01

    Full Text Available Abstract Background The leaves of globe artichoke and cultivated cardoon (Cynara cardunculus L. have significant pharmaceutical properties, which mainly result from their high content of polyphenolic compounds such as monocaffeoylquinic and dicaffeoylquinic acid (DCQ, and a range of flavonoid compounds. Results Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase (HQT encoding genes have been isolated from both globe artichoke and cultivated cardoon (GenBank accessions DQ915589 and DQ915590, respectively using CODEHOP and PCR-RACE. A phylogenetic analysis revealed that their sequences belong to one of the major acyltransferase groups (anthranilate N-hydroxycinnamoyl/benzoyltransferase. The heterologous expression of globe artichoke HQT in E. coli showed that this enzyme can catalyze the esterification of quinic acid with caffeoyl-CoA or p-coumaroyl-CoA to generate, respectively, chlorogenic acid (CGA and p-coumaroyl quinate. Real time PCR experiments demonstrated an increase in the expression level of HQT in UV-C treated leaves, and established a correlation between the synthesis of phenolic acids and protection against damage due to abiotic stress. The HQT gene, together with a gene encoding hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT previously isolated from globe artichoke, have been incorporated within the developing globe artichoke linkage maps. Conclusion A novel acyltransferase involved in the biosynthesis of CGA in globe artichoke has been isolated, characterized and mapped. This is a good basis for our effort to understand the genetic basis of phenylpropanoid (PP biosynthesis in C. cardunculus.

  11. Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids.

    Science.gov (United States)

    Gong, Yangmin; Wan, Xia; Jiang, Mulan; Hu, Chuanjiong; Hu, Hanhua; Huang, Fenghong

    2014-10-01

    Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have received growing attention due to their significant roles in human health. Currently the main source of these nutritionally and medically important fatty acids is marine fish, which has not met ever-increasing global demand. Microorganisms are an important alternative source also being explored. Although many microorganisms accumulate omega-3 LC-PUFAs naturally, metabolic engineering might still be necessary for significantly improving their yields. Here, we review recent research involving the engineering of microorganisms for production of omega-3 LC-PUFAs, including eicospentaenoic acid and docosohexaenoic acid. Both reconstitution of omega-3 LC-PUFA biosynthetic pathways and modification of existing pathways in microorganisms have demonstrated the potential to produce high levels of omega-3 LC-PUFAs. However, the yields of omega-3 LC-PUFAs in host systems have been substantially limited by potential metabolic bottlenecks, which might be caused partly by inefficient flux of fatty acid intermediates between the acyl-CoA and different lipid class pools. Although fatty acid flux in both native and heterologous microbial hosts might be controlled by several acyltransferases, evidence has suggested that genetic manipulation of one acyltransferase alone could significantly increase the accumulation of LC-PUFAs. The number of oleaginous microorganisms that can be genetically transformed is increasing, which will advance engineering efforts to maximize LC-PUFA yields in microbial strains.

  12. Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle.

    Science.gov (United States)

    Kazala, E Chris; Lozeman, Fred J; Mir, Priya S; Aalhus, Jennifer L; Schmutz, Sheila M; Weselake, Randall J

    2006-11-01

    Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14:0, r = 0.55, P muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r = 0.54, P muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA.

  13. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.

    Science.gov (United States)

    Yen, Chi-Liang Eric; Nelson, David W; Yen, Mei-I

    2015-03-01

    The intestine plays a prominent role in the biosynthesis of triacylglycerol (triglyceride; TAG). Digested dietary TAG is repackaged in the intestine to form the hydrophobic core of chylomicrons, which deliver metabolic fuels, essential fatty acids, and other lipid-soluble nutrients to the peripheral tissues. By controlling the flux of dietary fat into the circulation, intestinal TAG synthesis can greatly impact systemic metabolism. Genes encoding many of the enzymes involved in TAG synthesis have been identified. Among TAG synthesis enzymes, acyl-CoA:monoacylglycerol acyltransferase 2 and acyl-CoA:diacylglycerol acyltransferase (DGAT)1 are highly expressed in the intestine. Their physiological functions have been examined in the context of whole organisms using genetically engineered mice and, in the case of DGAT1, specific inhibitors. An emerging theme from recent findings is that limiting the rate of TAG synthesis in the intestine can modulate gut hormone secretion, lipid metabolism, and systemic energy balance. The underlying mechanisms and their implications for humans are yet to be explored. Pharmacological inhibition of TAG hydrolysis in the intestinal lumen has been employed to combat obesity and associated disorders with modest efficacy and unwanted side effects. The therapeutic potential of inhibiting specific enzymes involved in intestinal TAG synthesis warrants further investigation.

  14. Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process.

    Science.gov (United States)

    Merino, María C; Zamponi, Nahuel; Vranych, Cecilia V; Touz, María C; Rópolo, Andrea S

    2014-07-01

    Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

  15. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

    Directory of Open Access Journals (Sweden)

    Steffen Kawelke

    Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

  16. Deciphering Molecular Mechanism Underlying Hypolipidemic Activity of Echinocystic Acid

    Directory of Open Access Journals (Sweden)

    Li Han

    2014-01-01

    Full Text Available Our previous study showed that a triterpene mixture, consisting of echinocystic acid (EA and oleanolic acid (OA at a ratio of 4 : 1, dose-dependently ameliorated the hyperlipidemia and atherosclerosis in rabbits fed with high fat/high cholesterol diets. This study was aimed at exploring the mechanisms underlying antihyperlipidemic effect of EA. Molecular docking simulation of EA was performed using Molegro Virtual Docker (version: 4.3.0 to investigate the potential targets related to lipid metabolism. Based on the molecular docking information, isotope labeling method or spectrophotometry was applied to examine the effect of EA on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase, acyl-CoA:cholesterol acyltransferase (ACAT, and diacylglycerol acyltransferase (DGAT in rat liver microsomes. Our results revealed a strong affinity of EA towards ACAT and DGAT in molecular docking analysis, while low binding affinity existed between EA and HMG-CoA reductase as well as between EA and cholesteryl ester transfer protein. Consistent with the results of molecular docking, in vitro enzyme activity assays showed that EA inhibited ACAT and DGAT, with IC50 values of 103 and 139 μM, respectively, and exhibited no significant effect on HMG-CoA reductase activity. The present findings suggest that EA may exert hypolipidemic effect by inhibiting the activity of ACAT and DGAT.

  17. Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from Euonymus alatus (burning bush)

    Energy Technology Data Exchange (ETDEWEB)

    Durrett, Timothy; Ohlrogge, John; Pollard, Michael

    2016-05-03

    The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

  18. ASSOCIATIONS OF DGAT1 POLYMORPHISM WITH MILK CHARACTERISTICS IN SLOVAK DAIRY COWS

    Directory of Open Access Journals (Sweden)

    Katarína Vašíčková

    2013-02-01

    Full Text Available DGAT1 encodes diacylglycerol O-acyltransferase (EC 2.3.1.20, a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It was shown that the QTL variation is most likely caused by a nonconservative base substitution in the candidate gene DGAT1 changing lysine to alanine (K232A in the enzyme diacylglycerol O-acyltransferase (DGAT. In particular, the allele encoding the lysine 232 variant proved to be more efficient with regard to milk fat synthesis. The objective of the present study was to determine the allele frequencies and to verify the effects of the two DGAT variants (K232A polymorphism, A and K alleles on milk production traits in Slovak dairy cows. Samples of 196 dairy cows originating from 61 sires were genotyped for DGAT1 K232A polymorphism (A and K alleles using the PCR-RFLP technique. The frequencies of DGAT1 alleles were 0.88 (A and 0.12 (K. The performance data were collected during one season in order to minimize this effect. The overall milk yield (MILK, fat yield (FAT_Y, fat content (FAT_C, protein yield (PROT_Y, protein content (PROT_C and age at first calving (AGE1 were studied. The effect of DGAT1 polymorphism on fat and protein content in milk was confirmed. Further study is needed for explanation of effect of DGAT1 on the age at first calving.

  19. Allyl/propenyl phenol synthases from the creosote bush and engineering production of specialty/commodity chemicals, eugenol/isoeugenol, in Escherichia coli.

    Science.gov (United States)

    Kim, Sung-Jin; Vassão, Daniel G; Moinuddin, Syed G A; Bedgar, Diana L; Davin, Laurence B; Lewis, Norman G

    2014-01-01

    The creosote bush (Larrea tridentata) harbors members of the monolignol acyltransferase, allylphenol synthase, and propenylphenol synthase gene families, whose products together are able to catalyze distinct regiospecific conversions of various monolignols into their corresponding allyl- and propenyl-phenols, respectively. In this study, co-expression of a monolignol acyltransferase with either substrate versatile allylphenol or propenylphenol synthases in Escherichia coli established that various monolignol substrates were efficiently converted into their corresponding allyl/propenyl phenols, as well as providing proof of concept for efficacious conversion in a bacterial platform. This capability thus potentially provides an alternate source to these important plant phytochemicals, whether for flavor/fragrance and fine chemicals, or ultimately as commodities, e.g., for renewable energy or other intermediate chemical purposes. Previous reports had indicated that specific and highly conserved amino acid residues 84 (Phe or Val) and 87 (Ile or Tyr) of two highly homologous allyl/propenyl phenol synthases (circa 96% identity) from a Clarkia species mainly dictate their distinct regiospecific catalyzed conversions to afford either allyl- or propenyl-phenols, respectively. However, several other allyl/propenyl phenol synthase homologs isolated by us have established that the two corresponding amino acid 84 and 87 residues are not, in fact, conserved.

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

    Science.gov (United States)

    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.

  1. Molecular Cloning of a Novel Mouse Testis-specific Spermatogenic Cell Apoptosis Inhibitor Gene mTSARG7 as a Candidate Oncogene

    Institute of Scientific and Technical Information of China (English)

    Xiao-Jun TAN; Guang-Xiu LU; Xiao-Wei XING; Lu-Yun LI; Zhao-Di WU; Chang-Gao ZHONG; Dong-Song NIE; Jun-Jiang FU; Yang XIANG; Yun DENG

    2005-01-01

    A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

  2. Comprehensive Profiling of Proteome Changes Provide Insights of Industrial Penicillium chrysogenum During Pilot and Industrial Penicillin G Fermentation.

    Science.gov (United States)

    Cheng, Jing-Sheng; Zhao, Yan; Qiao, Bin; Lu, Hua; Chen, Yao; Yuan, Ying-Jin

    2016-07-01

    The intracellular proteomes of the Penicillium chrysogenum throughout pilot and industrial processes were investigated by using 2-DE combined with MALDI-TOF-TOF MS, respectively. We detected a total of 223 spots corresponding to 154 proteins and 231 spots corresponding to 157 proteins throughout pilot and industrial processes, respectively. The levels of glyceraldehyde-3-phosphate dehydrogenase increased (5.1- and 2.5-fold) under the pilot process, while its levels were no significant changes under the industrial process at 140 and 170 h when compared with that at 2 h. The levels of isocitrate lyase and fumarate hydratase were increased significantly under the industrial process, while their levels had no obvious changes after 20 h of fermentation throughout the pilot process. These results indicate that there were remarkable differences in carbohydrate metabolism (including glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid cycle) of P. chrysogenum during the pilot and industrial fermentations, which likely result in alterations of the primary metabolism and penicillin biosynthesis. Moreover, the differences in the levels of proteins involved in amino acid metabolisms (including valine, cysteine, and α-aminoadipic acid biosynthesis) indicated that the pilot and industrial processes influenced the supplies of penicillin precursors. Compared with that at 2 h, the maximum levels of superoxide (6.9-fold, at 32 h) and catalase (9-fold, at 80 h) during the industrial process and the maximum levels of superoxide (1.2-fold, at 20 h) and catalase (7.7-fold at 128 h) during the pilot process revealed the significant difference in cell redox homeostasis and stress responses during scale-up fermentation. Particularly, 10 spots corresponding to isopenicillin N synthetase and 4 spots corresponding to isopenicillin N (IPN) acyltransferase in pilot and industrial processes were identified, respectively. The levels of IPN acyltransferase (spots

  3. Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

    Directory of Open Access Journals (Sweden)

    Ashwin A. Raut

    2012-01-01

    Full Text Available Diacylglycerol O-acyltransferase 1 (DGAT1 is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.

  4. Identification of the Scopularide Biosynthetic Gene Cluster in Scopulariopsis brevicaulis

    Directory of Open Access Journals (Sweden)

    Mie Bech Lukassen

    2015-07-01

    Full Text Available Scopularide A is a promising potent anticancer lipopeptide isolated from a marine derived Scopulariopsis brevicaulis strain. The compound consists of a reduced carbon chain (3-hydroxy-methyldecanoyl attached to five amino acids (glycine, l-valine, d-leucine, l-alanine, and l-phenylalanine. Using the newly sequenced S. brevicaulis genome we were able to identify the putative biosynthetic gene cluster using genetic information from the structurally related emericellamide A from Aspergillus nidulans and W493-B from Fusarium pseudograminearum. The scopularide A gene cluster includes a nonribosomal peptide synthetase (NRPS1, a polyketide synthase (PKS2, a CoA ligase, an acyltransferase, and a transcription factor. Homologous recombination was low in S. brevicaulis so the local transcription factor was integrated randomly under a constitutive promoter, which led to a three to four-fold increase in scopularide A production. This indirectly verifies the identity of the proposed biosynthetic gene cluster.

  5. An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

    DEFF Research Database (Denmark)

    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco;

    2014-01-01

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary...... phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic...... and explain how cells switch neutral lipid metabolism from storage to consumption....

  6. Free radicals in tobacco smoke – analytical approach and biomedical significanc

    Directory of Open Access Journals (Sweden)

    Leon Kośmider

    2013-09-01

    Full Text Available Free radicals, i.e. atoms or groups of atoms containing one or more unpaired electrons, are significant constituents of tobacco smoke that contribute to its toxic properties. Radicals are generated during complex pyrolysis and combustion reactions in burning a cigarette cone. It has been shown that some free radicals found in tobacco smoke have relatively long half-time life (over 5 mins. We have reviewed modern analytical methods used for identification and quantitative analysis of free radicals in tobacco smoke, particularly the electron paramagnetic resonance combined with a spin-trapping approach. We also discussed the role of free radicals in etiology of respiratory and cardiovascular conditions among smokers. Finally, we reviewed biochemical mechanisms of various pathological conditions, including disturbances in lipid peroxidation, activity modification of lecithin-cholesterol acyltransferase and level high density lipoprotein, hyperactivity to substance P, and inactivation of neutral endopeptidase, that are thought to be contributed by free radicals from tobacco smoke.

  7. [Effect of protein-vitamin deficiency on the enzyme activity of lipolysis and the synthesis of cholesterol esters during hypokinesia].

    Science.gov (United States)

    Koshkenbaev, B Kh; Tazhibaev, Sh S; Maksimenko, V B; Sisemalieva, Zh S

    1985-01-01

    Balanced diet during 60-day hypokinesia leads to inhibition of lipoprotein lypase (LPLA) and liver triglyceride lypase (L-TGLA) activity of the rat blood serum. The level of very low density lipoproteins (VLDLP) grows, and suppression of lecithin-cholesteryl-acyltransferase (LCAT) activity is accompanied by reduction of the share of cholesterol derivatives with polyunsaturated fatty acids. Combined effects of protein-vitamin insufficiency and hypokinesia result in parversion of the lipolysis processes, that manifests in prevalence of L-TGLA over LPLA. The levels of VLDLP increase, and growth of LCAT activity is acompanied by the growth of cholesteryl linoleate share and level. Hypokinesia combined with the studied experimental diets was found to lead to increase of the free fatty acid level and to decrease of the blood serum levels of phospholipids and triglycerides.

  8. RNAi-mediated Ghrelin affects gastric H(+)-K(+)-ATPase activity and expression of GOAT-Ghrelin system in vitro.

    Science.gov (United States)

    Du, Gai M; Wu, Jie G; Luo, Bi P; Hu, Zhi H; Li, Liu A; Liu, Mao J

    2016-03-01

    Ghrelin has been implicated in the regulation of gastric functional development, and its physiological functions are mediated by Ghrelin-O-acyltransferase (GOAT) which is capable of generating the active form of this polypeptide hormone. However, whether and how ghrelin gene silencing may modify gastric acid secretion and GOAT-Ghrelin system is yet to be explored. The study was performed in gastric mucosal cells from weanling piglets in vitro. We evaluated the effect of ghrelin on gastric acid secretion, gene expression of GOAT and ghrelin as well as ghrelin levels by RNA interference assay. shGhrelin triggered the down-regulation of ghrelin mRNA expression (Pghrelin production and secretion (Pghrelin production and secretion were reduced in gastric mucosal cells and culture medium (Pghrelin gene achieved by RNAi-mediation inhibited the activity of H(+)-K(+)-ATPase and pepsin (PGhrelin gene inhibited the gastric acid secretion with decreased GOAT mRNA and acylated Ghrelin in gastric mucosal cells.

  9. Recent Advances in Potential Clinical Application of Ghrelin in Obesity

    Directory of Open Access Journals (Sweden)

    Christine Delporte

    2012-01-01

    Full Text Available Ghrelin is the natural ligand of the growth hormone secretagogue receptor (GHS-R1a. Ghrelin is a 28 amino acid peptide possessing a unique acylation on the serine in position 3 catalyzed by ghrelin O-acyltransferase (GOAT. Ghrelin stimulates growth hormone secretion, but also appetite, food intake, weight gain, and gastric emptying. Ghrelin is involved in weight regulation, obesity, type 2 diabetes, and metabolic syndrome. Furthermore, a better understanding of ghrelin biology led to the identification of molecular targets modulating ghrelin levels and/or its biological effects: GOAT, ghrelin, and GHS-R1a. Furthermore, a recent discovery, showing the involvement of bitter taste receptor T2R in ghrelin secretion and/or synthesis and food intake, suggested that T2R could represent an additional interesting molecular target. Several classes of ghrelin-related pharmacological tools for the treatment of obesity have been or could be developed to modulate the identified molecular targets.

  10. LCAT, HDL Cholesterol and Ischemic Cardiovascular Disease: A Mendelian Randomization Study of HDL Cholesterol in 54,500 Individuals

    DEFF Research Database (Denmark)

    Haase, Christiane L; Tybjærg-Hansen, Anne; Ali Qayyum, Abbas

    2012-01-01

    Background:Epidemiologically, high-density lipoprotein (HDL) cholesterol levels associate inversely with risk of ischemic cardiovascular disease. Whether this is a causal relation is unclear.Methods:We studied 10,281 participants in the Copenhagen City Heart Study (CCHS) and 50,523 participants...... in the Copenhagen General Population Study (CGPS), of which 991 and 1,693 participants, respectively, had developed myocardial infarction (MI) by August 2010. Participants in the CCHS were genotyped for all six variants identified by resequencing lecithin-cholesterol acyltransferase in 380 individuals. One variant......, S208T (rs4986970, allele frequency 4%), associated with HDL cholesterol levels in both the CCHS and the CGPS was used to study causality of HDL cholesterol using instrumental variable analysis.Results:Epidemiologically, in the CCHS, a 13% (0.21 mmol/liter) decrease in plasma HDL cholesterol levels...

  11. Identification and characterization of sebaceous gland atrophy-sparing DGAT1 inhibitors.

    Directory of Open Access Journals (Sweden)

    Eric S Muise

    Full Text Available Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1 has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting.

  12. Identification and Characterization of Sebaceous Gland Atrophy-Sparing DGAT1 Inhibitors

    Science.gov (United States)

    Muise, Eric S.; Zhu, Yonghua; Verras, Andreas; Karanam, Bindhu V.; Gorski, Judith; Weingarth, Drew; Lin, Hua V.; Hwa, Joyce; Thompson, John R.; Hu, Guanghui; Liu, Jian; He, Shuwen; DeVita, Robert J.; Shen, Dong-Ming; Pinto, Shirly

    2014-01-01

    Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting. PMID:24558447

  13. Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice.

    Science.gov (United States)

    Abraham, E; Bursten, S; Shenkar, R; Allbee, J; Tuder, R; Woodson, P; Guidot, D M; Rice, G; Singer, J W; Repine, J E

    1995-02-01

    Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.

  14. DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562-n

    Institute of Scientific and Technical Information of China (English)

    吕书晴; 许小平; 夏放; 居小萍; 李瑶; 应康; 毛裕民

    2003-01-01

    Objective: To study the molecular mechanism of different tumorigenicity in nude mice of human leukemia cell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells by using cDNA microarray technique. Results: Among the 12800 genes detected, some genes involved in material metabolism and material transport were differently expressed between K562-n and K562 cells. These genes include homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene, hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase, lysophosphatidic acid acyltransferase, alpha gene, argininosuccinate lyase gene, mitochondrial isocitrtate dehydrogenase, adhesion protein SQM1 gene, dimethylarginine dimethylamino-hydrolase gene, M1 subunit of ribonucleotide reductase and farnesyl pyrophosphate synthetase gene. Conclusion: The high tumorigenicity of K562-n cells is related to the different expression of some genes concerned with cell metabolism and material transpoert.

  15. A Review of Selected Genes with Known Effects on Performance and Health of Cattle

    Science.gov (United States)

    Casas, Eduardo; Kehrli, Marcus E.

    2016-01-01

    There are genetic conditions that influence production in dairy and beef cattle. The objective of this review was to describe relevant genetic conditions that have been associated with productivity and health in cattle. Genes or genomic regions that have been identified as a candidate for the condition will be included, and the genetic basis of the condition will be defined. Genes and genetic conditions included in this review are bovine leukocyte adhesion deficiency, deficiency of the uridine monophosphate synthase, bovine chronic interstitial nephritis, horn development, myostatin, complex vertebral malformation, leptin, osteopetrosis, apoptosis peptide activating factor 1, chondrodysplastic dwarfism, caseins, calpastatin, umbilical hernia, lactoglobulin, citrullinemia, cholesterol deficiency, prions, thyroglobulin, diacylglycerol acyltransferase, syndactyly, maple syrup urine disease, slick hair, Factor XI deficiency, and μ-Calpain. This review is not meant to be comprehensive, and relevant information is provided to ascertain genetic markers associated with the conditions. PMID:28018909

  16. A chloroplast pathway for the de novo biosynthesis of triacylglycerol in Chlamydomonas reinhardtii

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.; Xu, C.; Andre, C.

    2011-06-23

    Neutral lipid metabolism has been extensively studied in yeast, plants and mammals. In contrast, little information is available regarding the biochemical pathway, enzymes and regulatory factors involved in the biosynthesis of triacylglycerol (TAG) in microalgae. In the conventional TAG biosynthetic pathway widely accepted for yeast, plants and mammals, TAG is assembled in the endoplasmic reticulum (ER) from its immediate precursor diacylglycerol (DAG) made by ER-specific acyltransferases, and is deposited exclusively in lipid droplets in the cytosol. Here, we demonstrated that the unicellular microalga Chlamydomonas reinhardtii employs a distinct pathway that uses DAG derived almost exclusively from the chloroplast to produce TAG. This unique TAG biosynthesis pathway is largely dependent on de novo fatty acid synthesis, and the TAG formed in this pathway is stored in lipid droplets in both the chloroplast and the cytosol. These findings have wide implications for understanding TAG biosynthesis and storage and other areas of lipid metabolism in microalgae and other organisms.

  17. Detection of 1-O-malylglucose: pelargonidin 3-O-glucose-6''-O-malyltransferase activity in carnation (Dianthus caryophyllus).

    Science.gov (United States)

    Abe, Yutaka; Tera, Masayuki; Sasaki, Nobuhiro; Okamura, Masachika; Umemoto, Naoyuki; Momose, Masaki; Kawahara, Nobuo; Kamakura, Hiroyuki; Goda, Yukihiro; Nagasawa, Kazuo; Ozeki, Yoshihiro

    2008-09-05

    Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-beta-D-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.

  18. Diminished exercise capacity and mitochondrial bc1 complex deficiency in tafazzin-knockdown mice.

    Directory of Open Access Journals (Sweden)

    Corey ePowers

    2013-04-01

    Full Text Available The phospholipid, cardiolipin, is essential for maintaining mitochondrial structure and optimal function. Cardiolipin-deficiency in humans, Barth syndrome, is characterized by exercise intolerance, dilated cardiomyopathy, neutropenia and 3-methyl-glutaconic aciduria. The causative gene is the mitochondrial acyl-transferase, tafazzin that is essential for remodeling acyl chains of cardiolipin. We sought to determine metabolic rates in tafazzin-deficient mice during resting and exercise, and investigate the impact of cardiolipin deficiency on mitochondrial respiratory chain activities. Tafazzin knockdown in mice markedly impaired oxygen consumption rates during an exercise, without any significant effect on resting metabolic rates. CL-deficiency resulted in significant reduction of mitochondrial respiratory reserve capacity in neonatal cardiomyocytes that is likely to be caused by diminished activity of complex-III, which requires CL for its assembly and optimal activity. Our results may provide mechanistic insights of Barth syndrome pathogenesis.

  19. Biochemistry of Apple Aroma: A Review.

    Science.gov (United States)

    Espino-Díaz, Miguel; Sepúlveda, David Roberto; González-Aguilar, Gustavo; Olivas, Guadalupe I

    2016-12-01

    Flavour is a key quality attribute of apples defined by volatile aroma compounds. Biosynthesis of aroma compounds involves metabolic pathways in which the main precursors are fatty and amino acids, and the main products are aldehydes, alcohols and esters. Some enzymes are crucial in the production of volatile compounds, such as lipoxygenase, alcohol dehydrogenase, and alcohol acyltransferase. Composition and concentration of volatiles in apples may be altered by pre- and postharvest factors that cause a decline in apple flavour. Addition of biosynthetic precursors of volatile compounds may be a strategy to promote aroma production in apples. The present manuscript compiles information regarding the biosynthesis of volatile aroma compounds, including metabolic pathways, enzymes and substrates involved, factors that may affect their production and also includes a wide number of studies focused on the addition of biosynthetic precursors in their production.

  20. D38-cholesterol as a Raman active probe for imaging intracellular cholesterol storage

    Science.gov (United States)

    Alfonso-García, Alba; Pfisterer, Simon G.; Riezman, Howard; Ikonen, Elina; Potma, Eric O.

    2016-06-01

    We generated a highly deuterated cholesterol analog (D38-cholesterol) and demonstrated its use for selective vibrational imaging of cholesterol storage in mammalian cells. D38-cholesterol produces detectable signals in stimulated Raman scattering (SRS) imaging, is rapidly taken up by cells, and is efficiently metabolized by acyl-CoA cholesterol acyltransferase to form cholesteryl esters. Using hyperspectral SRS imaging of D38-cholesterol, we visualized cholesterol storage in lipid droplets. We found that some lipid droplets accumulated preferentially unesterified D38-cholesterol, whereas others stored D38-cholesteryl esters. In steroidogenic cells, D38-cholesteryl esters and triacylglycerols were partitioned into distinct sets of lipid droplets. Thus, hyperspectral SRS imaging of D38-cholesterol demonstrates a heterogeneous incorporation of neutral lipid species, i.e., free cholesterol, cholesteryl esters, and triacylglycerols, between individual lipid droplets in a cell.

  1. Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

    DEFF Research Database (Denmark)

    Holt, S.; J. Fowler, C.; Rocksén, D.;

    2004-01-01

    The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor a into the bronchoalveolar......-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent...... inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity. © 2004 Elsevier Inc. All rights reserved....

  2. Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

    Science.gov (United States)

    Nigam, Aeshna; Almabruk, Khaled H; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-07-25

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

  3. A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis.

    Directory of Open Access Journals (Sweden)

    Troco K Mihali

    Full Text Available Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.

  4. Memory and synaptic deficits in Hip14/DHHC17 knockout mice.

    Science.gov (United States)

    Milnerwood, Austen J; Parsons, Matthew P; Young, Fiona B; Singaraja, Roshni R; Franciosi, Sonia; Volta, Mattia; Bergeron, Sabrina; Hayden, Michael R; Raymond, Lynn A

    2013-12-10

    Palmitoylation of neurotransmitter receptors and associated scaffold proteins regulates their membrane association in a rapid, reversible, and activity-dependent fashion. This makes palmitoylation an attractive candidate as a key regulator of the fast, reversible, and activity-dependent insertion of synaptic proteins required during the induction and expression of long-term plasticity. Here we describe that the constitutive loss of huntingtin interacting protein 14 (Hip14, also known as DHHC17), a single member of the broad palmitoyl acyltransferase (PAT) family, produces marked alterations in synaptic function in varied brain regions and significantly impairs hippocampal memory and synaptic plasticity. The data presented suggest that, even though the substrate pool is overlapping for the 23 known PAT family members, the function of a single PAT has marked effects upon physiology and cognition. Moreover, an improved understanding of the role of PATs in synaptic modification and maintenance highlights a potential strategy for intervention against early cognitive impairments in neurodegenerative disease.

  5. Solving the puzzles of cutin and suberin polymer biosynthesis.

    Science.gov (United States)

    Beisson, Fred; Li-Beisson, Yonghua; Pollard, Mike

    2012-06-01

    Cutin and suberin are insoluble lipid polymers that provide critical barrier functions to the cell wall of certain plant tissues, including the epidermis, endodermis and periderm. Genes that are specific to the biosynthesis of cutins and/or aliphatic suberins have been identified, mainly in Arabidopsis thaliana. They notably encode acyltransferases, oxidases and transporters, which may have either well-defined or more debatable biochemical functions. However, despite these advances, important aspects of cutin and suberin synthesis remain obscure. Central questions include whether fatty acyl monomers or oligomers are exported, and the extent of extracellular assembly and attachment to the cell wall. These issues are reviewed. Greater emphasis on chemistry and biochemistry will be required to solve these unknowns and link structure with function.

  6. Plant cutin genesis: unanswered questions.

    Science.gov (United States)

    Domínguez, Eva; Heredia-Guerrero, José A; Heredia, Antonio

    2015-09-01

    The genesis of cutin, the main lipid polymer present in the biosphere, has remained elusive for many years. Recently, two main approaches have attempted to explain the process of cutin polymerization. One describes the existence of an acyltransferase cutin synthase enzyme that links activated monomers of cutin in the outer cell wall, while the other shows that plant cutin is the final result of an extracellular nonenzymatic self-assembly and polymerizing process of cutin monomers. In this opinion article, we explain both models and suggest that they could be pieces of a more complex biological scenario. We also highlight their different characteristics and current limitations, and suggest a potential synergism of both hypotheses.

  7. Building lipid barriers: biosynthesis of cutin and suberin.

    Science.gov (United States)

    Pollard, Mike; Beisson, Fred; Li, Yonghua; Ohlrogge, John B

    2008-05-01

    Cutin and suberin are the polymer matrices for lipophilic cell wall barriers. These barriers control the fluxes of gases, water and solutes, and also play roles in protecting plants from biotic and abiotic stresses and in controlling plant morphology. Although they are ubiquitous, cutin and suberin are the least understood of the major plant extracellular polymers. The use of forward and reverse genetic approaches in Arabidopsis has led to the identification of oxidoreductase and acyltransferase genes involved in the biosynthesis of these polymers. However, major questions about the underlying polymer structure, biochemistry, and intracellular versus extracellular assembly remain to be resolved. The analysis of plant lines with modified cutins and suberins has begun to reveal the inter-relationships between the composition and function of these polymers.

  8. Improving penicillin biosynthesis in Penicillium chrysogenum by glyoxalase overproduction.

    Science.gov (United States)

    Scheckhuber, Christian Q; Veenhuis, Marten; van der Klei, Ida J

    2013-07-01

    Genetic engineering of fungal cell factories mainly focuses on manipulating enzymes of the product pathway or primary metabolism. However, despite the use of strong promoters or strains containing the genes of interest in multiple copies, the desired strongly enhanced enzyme levels are often not obtained. Here we present a novel strategy to improve penicillin biosynthesis by Penicillium chrysogenum by reducing reactive and toxic metabolic by-products, 2-oxoaldehydes. This was achieved by overexpressing the genes encoding glyoxalase I and II, which resulted in a 10% increase in penicillin titers relative to the control strain. The protein levels of two key enzymes of penicillin biosynthesis, isopenicillin N synthase and isopenicillin N acyltransferase, were increased in the glyoxalase transformants, whereas their transcript levels remained unaltered. These results suggest that directed intracellular reduction of 2-oxoaldehydes prolongs the functional lifetime of these enzymes.

  9. Characterization of a spermidine hydroxycinnamoyltransferase in Malus domestica highlights the evolutionary conservation of trihydroxycinnamoyl spermidines in pollen coat of core Eudicotyledons.

    Science.gov (United States)

    Elejalde-Palmett, Carolina; de Bernonville, Thomas Dugé; Glevarec, Gaëlle; Pichon, Olivier; Papon, Nicolas; Courdavault, Vincent; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Lanoue, Arnaud; Besseau, Sébastien

    2015-12-01

    Phenolamides, so called hydroxycinnamic acid amides, are specialized metabolites produced in higher plants, involved in development, reproduction and serve as defence compounds in biotic interactions. Among them, trihydroxycinnamoyl spermidine derivatives were initially found to be synthetized by a spermidine hydroxycinnamoyltransferase (AtSHT) in Arabidopsis thaliana and to accumulate in the pollen coat. This study reports the identification, in Malus domestica, of an acyltransferase able to complement the sht mutant of Arabidopsis. The quantitative RT-PCR expression profile of MdSHT reveals a specific expression in flowers coordinated with anther development and tapetum cell activities. Three phenolamides including N (1),N (5),N (10)-tricoumaroyl spermidine and N (1),N (5)-dicoumaroyl-N (10)-caffeoyl spermidine identified by LC/MS, were shown to accumulate specifically in pollen grain coat of apple tree. Moreover, in vitro biochemical characterization confirmed MdSHT capacity to synthesize tri-substituted spermidine derivatives with a substrate specificity restricted to p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor. Further investigations of the presence of tri-substituted hydroxycinnamoyl spermidine conjugates in higher plants were performed by targeted metabolic analyses in pollens coupled with bioinformatic analyses of putative SHT orthologues in a wide range of available plant genomes. This work highlights a probable early evolutionary appearance in the common ancestral core Eudicotyledons of a novel enzyme from the BAHD acyltransferase superfamily, dedicated to the synthesis of trihydroxycinnamoyl spermidines in pollen coat. This pathway was maintained in most species; however, recent evolutionary divergences have appeared among Eudicotyledons, such as an organ reallocation of SHT gene expression in Fabales and a loss of SHT in Malvales and Cucurbitales.

  10. Intake of up to 3 Eggs per Day Is Associated with Changes in HDL Function and Increased Plasma Antioxidants in Healthy, Young Adults.

    Science.gov (United States)

    DiMarco, Diana M; Norris, Gregory H; Millar, Courtney L; Blesso, Christopher N; Fernandez, Maria Luz

    2017-03-01

    Background: HDL function may be more important than HDL concentration in determining risk for cardiovascular disease. In addition, HDL is a carrier of carotenoids and antioxidant enzymes, which protect HDL and LDL particles against oxidation.Objective: The goal of this study was to determine the impact of consuming 0-3 eggs/d on LDL and HDL particle size, HDL function, and plasma antioxidants in a young, healthy population.Methods: Thirty-eight healthy men and women [age 18-30 y, body mass index (in kg/m(2)) 18.5-29.9] participated in this 14-wk crossover intervention. Subjects underwent a 2-wk washout (0 eggs/d) followed by sequentially increasing intake of 1, 2, and 3 eggs/d for 4 wk each. After each period, fasting blood was collected for analysis of lipoprotein subfractions, plasma apolipoprotein (apo) concentration, lutein and zeaxanthin concentration, and activities of lecithin-cholesterol acyltransferase, cholesteryl ester transfer protein, and paraoxonase-1.Results: Compared with intake of 0 eggs/d, consuming 1-3 eggs/d resulted in increased large-LDL (21-37%) and large-HDL (6-13%) particle concentrations, plasma apoAI (9-15%), and lecithin-cholesterol acyltransferase activity (5-15%) (P HDL function and large-LDL particle concentration; however, intake of 2-3 eggs/d supported greater improvements in HDL function as well as increased plasma carotenoids. Overall, intake of ≤3 eggs/d favored a less atherogenic LDL particle profile, improved HDL function, and increased plasma antioxidants in young, healthy adults. This trial was registered at clinicaltrials.gov as NCT02531958.

  11. Reassessing the Potential Activities of Plant CGI-58 Protein.

    Science.gov (United States)

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  12. Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis.

    Science.gov (United States)

    van Erp, Harrie; Shockey, Jay; Zhang, Meng; Adhikari, Neil D; Browse, John

    2015-05-01

    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.

  13. Therapeutic potential of chalcones as cardiovascular agents.

    Science.gov (United States)

    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.

  14. The effects of sterol structure upon sterol esterification.

    Science.gov (United States)

    Lin, Don S; Steiner, Robert D; Merkens, Louise S; Pappu, Anuradha S; Connor, William E

    2010-01-01

    Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT(1) and ACAT(2) (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67% and in tissues by ACAT was 64%. Esterification of nine sterols (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined. The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82, respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74, respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29% and decreased the esterification by LCAT by 5.9%. Esterification by ACAT and LCAT was reduced, respectively, by 25 and 11% by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.

  15. Lipidomic and spatio-temporal imaging of fat by mass spectrometry in mice duodenum during lipid digestion.

    Science.gov (United States)

    Seyer, Alexandre; Cantiello, Michela; Bertrand-Michel, Justine; Roques, Véronique; Nauze, Michel; Bézirard, Valérie; Collet, Xavier; Touboul, David; Brunelle, Alain; 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.

  16. Reassessing the Potential Activities of Plant CGI-58 Protein.

    Directory of Open Access Journals (Sweden)

    Abdallah Khatib

    Full Text Available Comparative Gene Identification-58 (CGI-58 is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL, the initial enzyme responsible for the triacylglycerol (TAG catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  17. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

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    Atsushi eSato

    2014-09-01

    Full Text Available Triacylglycerol (TG synthesis is induced for energy and carbon storage in algal cells under nitrogen(N-starved conditions, and helps prevent reactive oxygen species production through fatty acid synthesis that consumes excessive reducing power. Here, the regulatory mechanism for the TG content in sulfur(S-starved cells of Chlamydomonas reinhardtii was examined, in comparison to that in N- or phosphorus(P-starved cells. S- and N-starved cells exhibited markedly increased TG contents with up-regulation of mRNA levels of diacylglycerol acyltransferase genes. S-Starvation also induced expression of the genes for phosphatidate synthesis. In contrast, P-starved cells exhibited little alteration of the TG content with almost no induction of these genes. The results implied deficient nutrient-specific regulation of the TG content. An arg9 disruptant defective in arginine synthesis, even without nutritional deficiencies, exhibited an increased TG content upon removal of supplemented arginine, which repressed protein synthesis. Repression of protein synthesis thus seemed crucial for TG accumulation in S- or N-starved cells. Meanwhile, the results of inhibitor experiments involving cells inferred that TG accumulation during S-starvation is supported by photosynthesis and de novo fatty acid synthesis. During S-starvation, sac1 and snrk2.2 disruptants, which are defective in the response to the ambient S-status, accumulated TG at lower and higher levels, respectively, than the wild type. The sac1 and snrk2.2 disruptants showed no or much greater up-regulation of diacylglycerol acyltransferase genes, respectively. In conclusion, TG synthesis would be activated in S-starved cells, through the diversion of metabolic carbon-flow from protein to TG synthesis, and simultaneously through up-regulation of the expression of a particular set of genes for TG synthesis at proper levels through the actions of SAC1 and SNRK2.2.

  18. Phosphatidylcholine formation by LPCAT1 is regulated by Ca2+ and the redox status of the cell

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    Soupene Eric

    2012-06-01

    Full Text Available Abstract Background Unsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A2. De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC from lysoPC and long-chain acyl-CoA. Results Activity of LPCAT1 is inhibited by Ca2+, and a Ca2+-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D392 and E403 to alanine rendered an enzyme insensitive to Ca2+, which established that Ca2+ binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents. Conclusion Mutant forms of LPCAT1 that are not inhibited by Ca2+ and sulfhydryl-alkylating and –oxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca2+ concentration of the cell.

  19. Paradoxical effects of increased expression of PGC-1α on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism

    Science.gov (United States)

    Choi, Cheol Soo; Befroy, Douglas E.; Codella, Roberto; Kim, Sheene; Reznick, Richard M.; Hwang, Yu-Jin; Liu, Zhen-Xiang; Lee, Hui-Young; Distefano, Alberto; Samuel, Varman T.; Zhang, Dongyan; Cline, Gary W.; Handschin, Christoph; Lin, Jiandie; Petersen, Kitt F.; Spiegelman, Bruce M.; Shulman, Gerald I.

    2008-01-01

    Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α has been shown to play critical roles in regulating mitochondria biogenesis, respiration, and muscle oxidative phenotype. Furthermore, reductions in the expression of PGC-1α in muscle have been implicated in the pathogenesis of type 2 diabetes. To determine the effect of increased muscle-specific PGC-1α expression on muscle mitochondrial function and glucose and lipid metabolism in vivo, we examined body composition, energy balance, and liver and muscle insulin sensitivity by hyperinsulinemic-euglycemic clamp studies and muscle energetics by using 31P magnetic resonance spectroscopy in transgenic mice. Increased expression of PGC-1α in muscle resulted in a 2.4-fold increase in mitochondrial density, which was associated with an ≈60% increase in the unidirectional rate of ATP synthesis. Surprisingly, there was no effect of increased muscle PGC-1α expression on whole-body energy expenditure, and PGC-1α transgenic mice were more prone to fat-induced insulin resistance because of decreased insulin-stimulated muscle glucose uptake. The reduced insulin-stimulated muscle glucose uptake could most likely be attributed to a relative increase in fatty acid delivery/triglyceride reesterfication, as reflected by increased expression of CD36, acyl-CoA:diacylglycerol acyltransferase1, and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase, that may have exceeded mitochondrial fatty acid oxidation, resulting in increased intracellular lipid accumulation and an increase in the membrane to cytosol diacylglycerol content. This, in turn, caused activation of PKCθ, decreased insulin signaling at the level of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and skeletal muscle insulin resistance. PMID:19066218

  20. Cloning and characterization of a gene involved in triacylglycerol biosynthesis and identification of additional homologous genes in the oleaginous bacterium Rhodococcus opacus PD630.

    Science.gov (United States)

    Alvarez, Adrian F; Alvarez, Héctor M; Kalscheuer, Rainer; Wältermann, Marc; Steinbüchel, Alexander

    2008-08-01

    The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc(2)155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant. RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88-99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.

  1. Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.

    Science.gov (United States)

    Sleight, R; Kent, C

    1980-11-25

    Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.

  2. Bovine gene polymorphisms related to fat deposition and meat tenderness

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    Marina R.S. Fortes

    2009-01-01

    Full Text Available Leptin, thyroglobulin and diacylglycerol O-acyltransferase play important roles in fat metabolism. Fat deposition has an influence on meat quality and consumers' choice. The aim of this study was to determine allele and genotype frequencies of polymorphisms of the bovine genes, which encode leptin (LEP, thyroglobulin (TG and diacylglycerol O-acyltransferase (DGAT1. A further objective was to establish the effects of these polymorphisms on meat characteristics. We genotyped 147 animals belonging to the Nelore (Bos indicus, Canchim (5/8 Bos taurus + 3/8 Bos indicus, Rubia Gallega X Nelore (1/2 Bos taurus + 1/2 Bos indicus, Brangus Three-way cross (9/16 Bos taurus + 7/16 Bos indicus and Braunvieh Three-way cross (3/4 Bos taurus + 1/4 Bos indicus breeds. Backfat thickness, total lipids, marbling score, ribeye area and shear force were fitted, using the General Linear Model (GLM procedure of the SAS software. The least square means of genotypes and genetic groups were compared using Tukey's test. Allele frequencies vary among the genetic groups, depending on Bos indicus versus Bos taurus influence. The LEP polymorphism segregates in pure Bos indicus Nelore animals, which is a new finding. The T allele of TG is fixed in Nelore, and DGAT1 segregates in all groups, but the frequency of allele A is lower in Nelore animals. The results showed no association between the genotypes and traits studied, but a genetic group effect on these traits was found. So, the genetic background remains relevant for fat deposition and meat tenderness, but the gene markers developed for Bos taurus may be insufficient for Bos indicus.

  3. Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

    Science.gov (United States)

    Schneider, Katja; Breuer, Christian; Kawamura, Ayako; Jikumaru, Yusuke; Hanada, Atsushi; Fujioka, Shozo; Ichikawa, Takanari; Kondou, Youichi; Matsui, Minami; Kamiya, Yuji; Yamaguchi, Shinjiro; Sugimoto, Keiko

    2012-01-01

    Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

  4. Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

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    Katja Schneider

    Full Text Available Brassinosteroids (BRs affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ, was partly rescued by supplying exogenous brassinolide (BL and castasterone (CS, indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY and 6-deoxocastasterone (6-deoxoCS, suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

  5. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes.

    Science.gov (United States)

    Priore, Paola; Siculella, Luisa; Gnoni, Gabriele Vincenzo

    2014-07-01

    Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

  6. 6-Gingerol Suppresses Adipocyte-Derived Mediators of Inflammation In Vitro and in High-Fat Diet-Induced Obese Zebra Fish.

    Science.gov (United States)

    Choi, Jia; Kim, Kui-Jin; Kim, Byung-Hak; Koh, Eun-Jeong; Seo, Min-Jung; Lee, Boo-Yong

    2017-02-01

    The present study was performed to investigate the molecular mechanism of 6-gingerol on adipocyte-mediated systemic inflammation in vitro and in high-fat diet-induced obese zebra fish. 6-Gingerol decreased adipogenesis due to the suppression of adipocyte differentiation markers, including peroxisome proliferator-activated receptor gamma, CCAATT enhancer binding protein α, and adipocyte protein 2, and triglyceride synthesis enzymes, including sterol regulatory element-binding protein-1, fatty acid synthase, lysophosphatidic acid acyltransferase, and acyl-coA : diacylglycerol acyltransferase 1, in 3T3-L1. A coculture insert system using 3T3-L1 with RAW 264.7 (coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages) revealed that 6-gingerol increased anti-inflammatory cytokine interleukin-10. The expression of TNFα, monocyte chemotactic protein-1, interleukin-1β, and interleukin-6 were decreased in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol. Moreover, the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages treated with 6-gingerol inhibited the protein expression of TNFα and monocyte chemotactic protein-1 in RAW 264.7. 6-Gingerol decreased c-JUN N-terminal kinase and I kappa B kinase beta and its downstream target AP-1 expression in the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages. Furthermore, 6-gingerol decreased the expression of inducible nitric oxide synthase stimulated by the coculture insert system using fully differentiated 3T3-L1 cells with RAW 264.7 macrophages in RAW 264.7 and attenuated nitric oxide production in diet-induced obese zebra fish. Our results suggest that 6-gingerol suppresses inflammation through the regulation of the c-JUN N-terminal kinase-I kappa B kinase beta and its downstream targets.

  7. Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

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    Bovenberg Roel AL

    2009-05-01

    Full Text Available Abstract Background Penicillium chrysogenum converts isopenicillin N (IPN into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT, which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like. We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway. Results The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null ial mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic and amidohydrolase (6-APA forming activities in vivo. When the canonical ARL motif (leading to peroxisomal targeting was added to the C-terminus of the IAL protein (IALARL by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in Escherichia coli. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive in vitro. Conclusion No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the P. chrysogenum IAL, the A. nidulans IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the ial gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in A. nidulans, the ial and penDE genes might have been formed from a single

  8. Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

    Science.gov (United States)

    Alban, C; Joyard, J; Douce, R

    1989-05-01

    The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3

  9. Sex Differences in Long Chain Fatty Acid Utilization and Fatty Acid Binding Protein Concentration in Rat Liver

    Science.gov (United States)

    Ockner, Robert K.; Burnett, David A.; Lysenko, Nina; Manning, Joan A.

    1979-01-01

    Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [14C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more 14C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [14C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [14C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [14C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified. The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to

  10. 小肠胆固醇吸收相关蛋白的研究进展%Several proteins involved in absorption of cholesterol in small intestine

    Institute of Scientific and Technical Information of China (English)

    袁敏; 徐东刚

    2015-01-01

    多种蛋白参与了小肠胆固醇的吸收,其中尼曼-匹克C1型类似蛋白1(Niemann-Pick C1 like 1,NPC1L1)主要介导小肠对胆固醇的吸收;小肠吸收的游离胆固醇在酰基辅酶A-胆固醇酰基转移酶2[acyl-coenzyme A(CoA)∶cholesterol acyltransferase 2,ACAT2]的催化下形成胆固醇酯并经淋巴系统进入血液循环,而未被酯化的胆固醇则通过ATP结合盒转运蛋白G5/G8[ATP-binding cassette(ABC) transporters G5/G8,ABCG5/ABCG8]分泌入肠腔,转录因子肝X受体( liver X receptor,LXR)在小肠胆固醇吸收过程中发挥了重要的调节作用。该文主要对小肠胆固醇吸收相关蛋白NPC1 L1、ABCG5/ABCG8、ACAT2和LXR的研究进展进行了综述。%Several proteins are involved in the absorption of cholesterol in small intestine.Niemann-Pick C1 like 1 (NPC1L1) mainly mediates the absorption of cholesterol, and acyl-coenzyme A ( CoA)∶cholesterol acyltransferase 2 (ACAT2) catalyzes the free cholesterol absorpted by intestine into cholesterol ester,while unesterified free cholesterol is secreted into intestinal lumen by ATP-binding cassette(ABC) transporters G5/G8(ABCG5/ABCG8).Transcription factor liver X receptor( LXR) plays an important role in the process of intestinal cholesterol absorption.The research progress in NPC1L1,ABCG5/ABCG8,ACAT2 and LXR is reviewed in this article.

  11. Adsorption of lipoproteins onto mineral dust surfaces: a possible factor in the pathogenesis of particle-induced pulmonary fibrosis?

    Science.gov (United States)

    Bogatu, Bettina; Contag, Bodo

    2005-01-01

    We compare the adsorption behavior of high density lipoproteins (HDL) and low density lipoproteins (LDL) on "fibrogenic" and "nonfibrogenic" mineral dusts. The adsorption tests with bovine lipoprotein concentrate and human serum produced the following results: 1) All seven examined fibrogenic dusts (SiO2 DQ12, SiO2 F600, silica, graphite, TiC, kaolin, talc) adsorbed significantly more high density lipoproteins (HDL), than the five examined nonfibrogenic (inert) dusts (TiO2, SnO2, Al2O3, Fe2O3, Fe3O4). This different behavior was particularly conspicuous in the presence of competing adsorbates (serum proteins). 2) In contrast, the adsorption of LDL did not correlate with the fibrogenicity of the mineral dusts. 3) The known silicosis-protective substance polyvinylpyridine-N-oxide inhibits the HDL adsorption of alpha-quartz. These results indicate that the adsorption of HDL could have a causal relationship with the triggering of a fibrotic reaction. The adsorption on the surface of fibrogenic dust particles provides an exceptional opportunity for the intake of HDL by macrophages. During the phagocytosis of the inhaled dust particles, the HDL adsorbed on the surface of the particles could be taken up by macrophages regardless of the receptor. There the HDL particles and/or compounds associated with them, such as lecithin-cholesterol-acyltransferase, could stimulate the macrophages to release fibrogenic mediators by some yet unknown mechanism.

  12. Comparison of cardiolipins from Drosophila strains with mutations in putative remodeling enzymes.

    Science.gov (United States)

    Schlame, Michael; Blais, Steven; Edelman-Novemsky, Irit; Xu, Yang; Montecillo, Fleurise; Phoon, Colin K L; Ren, Mindong; Neubert, Thomas A

    2012-07-01

    Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A(2) had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.

  13. Incorporation of extracellular fatty acids by a fatty acid kinase-dependent pathway in Staphylococcus aureus.

    Science.gov (United States)

    Parsons, Joshua B; Frank, Matthew W; Jackson, Pamela; Subramanian, Chitra; Rock, Charles O

    2014-04-01

    Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4 ) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

  14. Metabolism of 2-acetylaminofluorene by Shasta rainbow trout

    Energy Technology Data Exchange (ETDEWEB)

    Sikka, H.C.; Elmarakaby, S.A.; Steward, R.; Maslanka, R.; Shappell, N.; Kumar, S. [State University of New York, Buffalo, NY (United States); Devanaboyina, U.; Gupta, R.C. [Univ. of Kentucky, Lexington, KY (United States)

    1994-12-31

    In contrast to several mammalian species, Shasta strain rainbow trout (Oncorhynchus mykiss), is resistant to the hepatocarcinogenic effects of 2-acetylaminofluorene (AAF). In order to understand the mechanism underlying this resistance, the authors have investigated the in vitro metabolism of AAF by trout liver and examined the formation of AAF-DNA adducts in the liver of Shasta trout treated with AAF. The major AAF metabolites produced by trout liver microsomes were 7-hydroxy-AAF and 5-hydroxy-AAF which accounted for more than 95% of the total AAF metabolites. N-hydroxy-AAF was a minor metabolite representing about 1% of total AAF metabolites. The levels of N-hydroxy-AAF sulfotransferase and N-hydroxy-AAF acyltransferase, the cytosolic enzymes implicated in the metabolic activation of N-hydroxy-AAF to reactive intermediates capable of binding to cellular macromolecules were extremely low in trout liver. AAF exhibited a low degree of binding to the liver DNA of trout treated with 15 mg AAF/kg body wt. The total AAF-DNA adduct level reached a maximum 24 hours after treatment, persisted until 11 days and declined to nearly 20% of the maximum level after 18 days. N-deoxyguanosin-8-yl-2-aminofluorene was the major AAF-DNA adduct in the trout liver. The ability of trout liver to form relatively large amounts of detoxification products of AAF with little formation of activation products may partly explain the resistance of Shasta trout to AAF-induced hepatocarcinogenesis.

  15. Participation of ghrelin signalling in the reciprocal regulation of hypothalamic NPY/POMC-mediated appetite control in amphetamine-treated rats.

    Science.gov (United States)

    Yu, Ching-Han; Chu, Shu-Chen; Chen, Pei-Ni; Hsieh, Yih-Shou; Kuo, Dong-Yih

    2017-02-14

    Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) have been documented to participate in amphetamine (AMPH)-induced appetite suppression. This study investigated whether ghrelin signalling is associated with changes in NPY/POMC-mediated appetite control. Rats were given AMPH daily for four days, and changes in food intake, body weight, plasma ghrelin, hypothalamic NPY, melanocortin 3 receptor (MC3R), ghrelin O-acyltransferase (GOAT), acyl ghrelin (AG) and ghrelin receptor (GHSR1a) were examined and compared. Food intake, body weight and NPY expression decreased, while MC3R expression increased and expressed reciprocally to NPY expression during AMPH treatment. Plasma ghrelin and hypothalamic AG/GOAT/GHSR1a expression decreased on Day 1 and Day 2, which was associated with the positive energy metabolism, and returned to normal levels on Day 3 and Day 4, which was associated with the negative energy metabolism; this expression pattern was similar to that of NPY. Infusion with a GHSR1a antagonist or an NPY antisense into the brain enhanced the decrease in NPY and AG/GOAT/GHSR1a expression and the increase in MC3R expression compared to the AMPH-treated group. Peripheral ghrelin and the central ghrelin system participated in the regulation in AMPH-induced appetite control. These results shed light on the involvement of ghrelin signalling in reciprocal regulation of NPY/POMC-mediated appetite control and may prove useful for the development of anti-obesity drugs.

  16. Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

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    Lin Yao

    2016-06-01

    Full Text Available Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp and pksT-2 (7901 bp suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

  17. Ghrelin and the brain-gut axis as a pharmacological target for appetite control.

    Science.gov (United States)

    Seim, Inge; El-Salhy, Magdy; Hausken, Trygve; Gundersen, Doris; Chopin, Lisa

    2012-01-01

    Appetite regulation is highly complex and involves a large number of orexigenic and anorexigenic peptide hormones. These are small, processed, secreted peptides derived from larger prepropeptide precursors. These peptides are important targets for the development of therapeutics for obesity, a global health epidemic. As a case study, we consider the ghrelin axis. The ghrelin axis is likely to be a particularly useful drug target, as it also plays a role in energy homeostasis, adipogenesis, insulin regulation and reward associated with food intake. Ghrelin is the only known circulating gut orexigenic peptide hormone. As it appears to play a role in diet-induced obesity, blocking the action of ghrelin is likely to be effective for treating and preventing obesity. The ghrelin peptide has been targeted using a number of approaches, with ghrelin mirror-image oligonucleotides (Spiegelmers) and immunotherapy showing some promise. The ghrelin receptor, the growth hormone secretagogue receptor, may also provide a useful target and a number of antagonists and inverse agonists have been developed. A particularly promising new target is the enzyme which octanoylates ghrelin, ghrelin O-acyltransferase (GOAT), and drugs that inhibit GOAT are likely to circumvent pharmacological issues associated with approaches that directly target ghrelin or its receptor.

  18. Lipid characterization of an arachidonic acid-rich oil producing fungus Mortierella alpina

    Institute of Scientific and Technical Information of China (English)

    Wenjia Wu; Jiacheng Yan; Xiaojun Ji; Xin Zhang; Jingsheng Shang; Lina Sun; Lujing Ren; He Huang

    2015-01-01

    Mortierel a alpina has been considered as the most effective producer of arachidonic acid (ARA)-rich oil. It was found that several methods could improve the percentage of ARA in total lipids successful y, as they activated the desaturation system on the endoplasmic reticulum. Additionally, in M. alpina the ARA exists in several forms, such as triacylglycerol (TAG), and diacylglycerol (DAG). These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil. However, few works revealed de-tailed fatty acid distribution among lipid classes, which to some extent impeded the accurate regulation in ARA accumulation. Herein, this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M. alpina. The result dem-onstrates that TAG was the dominant component of the total lipids, and it is the main form for ARA storage. The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady. Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modification of sat-urated and monounsaturated fatty acids in other lipid classes. This work could be helpful for further optimization of ARA-rich TAG production.

  19. Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed.

    Science.gov (United States)

    Metz, J G; Pollard, M R; Anderson, L; Hayes, T R; Lassner, M W

    2000-03-01

    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.

  20. Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis.

    Science.gov (United States)

    Lardizabal, K D; Metz, J G; Sakamoto, T; Hutton, W C; Pollard, M R; Lassner, M W

    2000-03-01

    Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.

  1. In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats

    Directory of Open Access Journals (Sweden)

    Pei Lin

    2014-01-01

    Full Text Available Mechanisms of the water extracts of Polygoni Multiflori Radix (PMR and its processed products (PMRP on liver lipid metabolism were observed in this paper. Aqueous extract of PMR and PMRP was given to nonalcoholic fatty liver model rats, respectively. PMR was better in reducing the contents of very low density lipoprotein (VLDL than PMRP and the positive control groups. In the aspect of regulating TG, medium dose PMR reduced the activity of diacylglycerol acyltransferase (DGAT to 1536±47.69 pg/mL (P<0.001 and promoted the expression of hepatic lipase (HL to 23.59±0.2758 U/mL (P<0.05. HL promotion ability of medium dose PMR was similar with the simvastatin positive control. Both medium and high dose of PMR showed significant alterations in TC, which were related to the downregulation effects on hydroxyl methyl-glutaryl coenzyme A reductase (HMGCR and upregulation effects on cholesterol 7-alpha-hydroxylase or cytochrome P450 7A (CYP7A. Quantitative relationships research indicated that the prominent effect on inhibiting the content of HMGCR (r=0.756, P<0.05 was strongly positive correlated with to the TC regulation effects. Effects of PMR on enhancing decomposition rate or reducing de novo synthesis rate of TG and TC were better than PMRP.

  2. The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly.

    Science.gov (United States)

    Lashbrooke, Justin; Adato, Avital; Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Granell, Antonio; Costa, Fabrizio; Aharoni, Asaph

    2015-12-01

    The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.

  3. Association between Single Nucleotide Polymorphism rs1044925 and the Risk of Coronary Artery Disease and Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Dong-Feng Wu

    2014-02-01

    Full Text Available The present study was performed to clarify the association between the acyl-CoA:cholesterol acyltransferase-1 (ACAT-1 single nucleotide polymorphism (SNP rs1044925 and the risk of coronary artery disease (CAD and ischemic stroke (IS in the Guangxi Han population. Polymerase chain reaction and restriction fragment length polymorphism was performed to determine the genotypes of the ACAT-1 SNP rs1044925 in 1730 unrelated subjects (CAD, 587; IS, 555; and healthy controls; 588. The genotypic and allelic frequencies of rs1044925 were significantly different between the CAD patients and controls (p = 0.015 and borderline different between the IS patients and controls (p = 0.05. The AC/CC genotypes and C allele were associated with a decreased risk of CAD and IS (CAD: p = 0.014 for AC/CC vs. AA, p = 0.022 for C vs. A; IS: p = 0.014 for AC/CC vs. AA; p = 0.017 for C vs. A. The AC/CC genotypes in the healthy controls, but not in CAD or IS patients, were associated with an increased serum high-density lipoprotein cholesterol (HDL-C concentration. The present study shows that the C allele carriers of ACAT-1 rs1044925 were associated with an increased serum HDL-C level in the healthy controls and decreased risk in CAD and IS patients.

  4. A culture independent method for the detection of Aeromonas sp. from water samples

    Directory of Open Access Journals (Sweden)

    Fadua Latif-Eugenín

    2016-01-01

    Full Text Available The genus Aeromonas is present in a wide variety of water environments and is recognised as potentially pathogenic to humans and animals. Members of this genus are often confused with Vibrio when using automated, commercial identification systems that are culture-dependent. This study describes a polymerase chain reaction (PCR detection method for Aeromonas that is culture- independent and that targets the glycerophospholopid-cholesterol acyltransferase (gcat gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCATPCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 μL to 4 mL. The proposed molecular method is much faster (5 or 29 h than the culturing method (24 or 48 h whether performed directly or after a pre-enrichment step and it will enable the fast detection of Aeromonas in water samples helping to prevent a possible transmission to humans.

  5. Characterization of the microbial community and polyketide biosynthetic potential in the palmerolide-producing tunicate Synoicum adareanum.

    Science.gov (United States)

    Riesenfeld, Christian S; Murray, Alison E; Baker, Bill J

    2008-11-01

    Palmerolide A (1) is a macrolide isolated from the Antarctic tunicate Synoicum adareanum that is of interest due to its potential as an antimelanoma drug. Biosynthesis is predicted to occur via a hybrid PKS-NRPS pathway within S. adareanum, but the identity of the palmerolide-producing organism (host or putative host-associated microorganism) has not been established. Microscopic observation revealed a dense microbial community inside the tunicate, and evidence from 16S rRNA gene DGGE profiles and clone library sequences suggests that the bacterial community has moderate phylogenetic complexity. The alpha and gamma classes of Proteobacteria account for ∼75% of the cloned 16S rRNA genes, and the majority of these sequences are affiliated with the genera Pseudovibrio and Microbulbifer. DNA sequences encoding type I PKS ketosynthase (KS) domains were detected by PCR. The S. adareanum KS sequences, which affiliate with the trans-AT clade, are similar to portions of PKS proteins that lack integrated acyltransferase domains in pathways for generating bioactive polyketide compounds, including bryostatin, leinamycin, and pederin.

  6. Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

    Directory of Open Access Journals (Sweden)

    Cresten B Mansfeldt

    Full Text Available Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596 of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

  7. Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

    Science.gov (United States)

    Mansfeldt, Cresten B; Richter, Lubna V; Ahner, Beth A; Cochlan, William P; Richardson, Ruth E

    2016-01-01

    Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

  8. Intestine-targeted DGAT1 inhibition improves obesity and insulin resistance without skin aberrations in mice.

    Directory of Open Access Journals (Sweden)

    Naoto Tsuda

    Full Text Available OBJECTIVE: Diacylglycerol O-acyltransferase 1 (DGAT1 catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice. DESIGN AND METHODS: We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500, reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO mice. RESULTS: The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice. CONCLUSIONS: Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance.

  9. Phylogeny of type I polyketide synthases (PKSs) in fungal entomopathogens and expression analysis of PKS genes in Beauveria bassiana BCC 2660.

    Science.gov (United States)

    Punya, Juntira; Swangmaneecharern, Pratchya; Pinsupa, Suparat; Nitistaporn, Pornpen; Phonghanpot, Suranat; Kunathigan, Viyada; Cheevadhanarak, Supapon; Tanticharoen, Morakot; Amnuaykanjanasin, Alongkorn

    2015-06-01

    Entomopathogenic fungi are able to invade and kill insects. Various secondary metabolites can mediate the interaction of a fungal pathogen with an insect host and also help the fungus compete with other microbes. Here we screened 23 isolates of entomopathogenic fungi for polyketide synthase (PKS) genes and amplified 72 PKS gene fragments using degenerate PCR. We performed a phylogenetic analysis of conserved ketosynthase and acyltransferase regions in these 72 sequences and 72 PKSs identified from four insect fungal genome sequences. The resulting genealogy indicated 47 orthologous groups with 99-100 % bootstrap support, suggesting shared biosynthesis of identical or closely related compounds from different fungi. Three insect-specific groups were identified among the PKSs in reducing clades IIa, IIb, and III, which comprised PKSs from 12, 9, and 30 fungal isolates, respectively. A IIa-IIb pair could be found in seven fungi. Expression analyses revealed that eleven out of twelve PKS genes identified in Beauveria bassiana BCC 2660 were expressed in culture. PKS genes from insect-specific clades IIa and IIb were expressed only in insect-containing medium, while others were expressed only in PDB or in CYB, PDB and SDY. The data suggest the potential production of several polyketides in culture.

  10. Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain

    DEFF Research Database (Denmark)

    Moesgaard, B.; Petersen, G.; Hansen, Harald S.;

    2000-01-01

    of various age (1, 6, 12, 19, 30, and ~70 days) by the use of P NMR spectroscopy of lipid extracts. This ability to accumulate NAPE was compared with the activity of N-acyltransferase and of NAPE-hydrolyzing phospholipase D (NAPE-PLD) in brain microsomes. These two enzymes are involved in the formation...... and degradation of NAPE, respectively. The results showed that 1) the ability to accumulate NAPE during post-decapitative ischemia is especially high in the youngest rats and is markedly reduced in older brains [in 1-day-old rat brains NAPE accumulated to 1.5% of total phospholipids, while in 30-day-old rat......N-acyl-ethanolamine phospholipids (NAPE) can be formed as a stress response during neuronal injury, and they are precursors for N-acyl- ethanolamines (NAE), some of which are endocannabinoids. The levels of NAPE accumulated during post-decapitative ischemia (6 h at 37°C) were studied in rat brains...

  11. Piperine potentiates the hypocholesterolemic effect of curcumin in rats fed on a high fat diet.

    Science.gov (United States)

    Tu, Yaosheng; Sun, Dongmei; Zeng, Xiaohui; Yao, Nan; Huang, Xuejun; Huang, Dane; Chen, Yuxing

    2014-07-01

    It has previously been demonstrated that curcumin possesses a hypocholesterolemic effect and potentiates numerous pharmacological effects of curcumin, however, the mechanisms underlying this hypocholesterolemic effect and the interaction between curcumin and piperine remain to be elucidated. In the present study, male Sprague-Dawley rats were fed on a high-fat diet (HFD) to establish a hyperlipidemia (HLP) model. Co-administration of curcumin plus piperine was found to decrease the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol in the serum and liver, as well as increase the levels of fecal TC, TG and total bile acid, compared with administration of curcumin alone. Curcumin plus piperine also markedly increased the levels of high-density lipoprotein cholesterol. Furthermore, compared with administration of curcumin alone, administration of curcumin plus piperine resulted in a significant upregulation of the activity and gene expression of apolipoprotein AI (ApoAI), lecithin cholesterol acyltransferase (LCAT), cholesterol 7α-hydroxylase (CYP7A1) and low-density lipoprotein receptor (LDLR). In conclusion, these results indicated that co-administration of curcumin plus piperine potentiates the hypocholesterolemic effects of curcumin by increasing the activity and gene expression of ApoAI, CYP7A1, LCAT and LDLR, providing a promising combination for the treatment of HLP.

  12. Beneficial effects of curcumin on hyperlipidemia and insulin resistance in high-fat-fed hamsters.

    Science.gov (United States)

    Jang, Eun-Mi; Choi, Myung-Sook; Jung, Un Ju; Kim, Myung-Joo; Kim, Hye-Jin; Jeon, Seon-Min; Shin, Su-Kyung; Seong, Chi-Nam; Lee, Mi-Kyung

    2008-11-01

    This study investigated the effect of curcumin (0.05-g/100-g diet) supplementation on a high-fat diet (10% coconut oil, 0.2% cholesterol, wt/wt) fed to hamsters, one of the rodent species that are most closely related to humans in lipid metabolism. Curcumin significantly lowered the levels of free fatty acid, total cholesterol, triglyceride, and leptin and the homeostasis model assessment of insulin resistance index, whereas it elevated the levels of high-density lipoprotein cholesterol and apolipoprotein (apo) A-I and paraoxonase activity in plasma, compared with the control group. The levels of hepatic cholesterol and triglyceride were also lower in the curcumin group than in the control group. In the liver, fatty acid beta-oxidation activity was significantly higher in the curcumin group than in the control group, whereas fatty acid synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and acyl coenzyme A:cholesterol acyltransferase activities were significantly lower. Curcumin significantly lowered the lipid peroxide levels in the erythrocyte and liver compared with the control group. These results indicate that curcumin exhibits an obvious hypolipidemic effect by increasing plasma paraoxonase activity, ratios of high-density lipoprotein cholesterol to total cholesterol and of apo A-I to apo B, and hepatic fatty acid oxidation activity with simultaneous inhibition of hepatic fatty acid and cholesterol biosynthesis in high-fat-fed hamsters.

  13. Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase.

    Science.gov (United States)

    Petković, Hrvoje; Sandmann, Axel; Challis, Iain R; Hecht, Hans-Jürgen; Silakowski, Barbara; Low, Lindsey; Beeston, Nicola; Kuscer, Enej; Garcia-Bernardo, Jose; Leadlay, Peter F; Kendrew, Steven G; Wilkinson, Barrie; Müller, Rolf

    2008-02-07

    The production of epothilone mixtures is a direct consequence of the substrate tolerance of the module 3 acyltransferase (AT) domain of the epothilone polyketide synthase (PKS) which utilises both malonyl- and methylmalonyl-CoA extender units. Particular amino acid motifs in the active site of AT domains influence substrate selection for methylmalonyl-CoA (YASH) or malonyl-CoA (HAFH). This motif appears in hybrid form (HASH) in epoAT3 and may represent the molecular basis for the relaxed specificity of the domain. To investigate this possibility the AT domains from modules 2 and 3 of the epothilone PKS were examined in the heterologous DEBS1-TE model PKS. Substitution of AT1 of DEBS1-TE by epoAT2 and epoAT3 both resulted in functional PKSs, although lower yields of total products were observed when compared to DEBS1-TE (2% and 11.5% respectively). As expected, epoAT3 was significantly more promiscuous in keeping with its nature during epothilone biosynthesis. When the mixed motif (HASH) of epoAT3 within the hybrid PKS was mutated to HAFH (indicative of malonyl-CoA selection) it resulted in a non-productive PKS. When this mixed motif was converted to YASH (indicative of methylmalonyl-CoA selection) the selectivity of the hybrid PKS for methylmalonyl-CoA showed no statistically significant increase, and was associated with a loss of productivity.

  14. Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus)

    Science.gov (United States)

    Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Delgado, María Jesús; Valenciano, Ana Isabel; Unniappan, Suraj

    2017-01-01

    Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish. PMID:28338019

  15. Fumonisins--mycotoxins produced by Fusarium moniliforme.

    Science.gov (United States)

    Norred, W P

    1993-03-01

    Fumonisins are toxic metabolites of the fungus Fusarium moniliforme, which is a common contaminant of corn everywhere in the world. The fumonisins are carcinogenic in laboratory rats, and cause acute toxicity of domestic animals that mimics field cases of disease attributed to contamination of feed by F. moniliforme. These include both equine leukoencephalomalacia and porcine pulmonary edema. Fusarium moniliforme contamination of corn consumed by humans in certain areas of the world is associated with higher than average incidence of esophageal cancer, and fumonisins may be responsible. Analytical methods have been developed for fumonisins, but improvements are needed so that more accurate, less expensive, and more rapid assays of food and feedstuffs can be done. Fumonisins are structurally similar to sphingosine, and may exert their biological activity through their ability to block key enzymes (sphinganine- and sphingosine-N-acyltransferases) involved in sphingolipid biosynthesis. Much more research is needed to define the extent to which this mycotoxin adversely affects the food supply, and its involvement in animal and human diseases.

  16. Triterpenic Acids Present in Hawthorn Lower Plasma Cholesterol by Inhibiting Intestinal ACAT Activity in Hamsters

    Directory of Open Access Journals (Sweden)

    Yuguang Lin

    2011-01-01

    Full Text Available Hawthorn (Crataegus pinnatifida is an edible fruit used in traditional Chinese medicine to lower plasma lipids. This study explored lipid-lowering compounds and underlying mechanisms of action of hawthorn. Hawthorn powder extracts inhibited acylCoA:cholesterol acyltransferase (ACAT activity in Caco-2 cells. The inhibitory activity was positively associated with triterpenic acid (i.e., oleanolic acid (OA and ursolic acid (UA contents in the extracts. Cholesterol lowering effects of hawthorn and its potential additive effect in combination with plant sterol esters (PSE were further studied in hamsters. Animals were fed a semi-synthetic diet containing 0.08% (w/w cholesterol (control or the same diet supplemented with (i 0.37% hawthorn dichloromethane extract, (ii 0.24% PSE, (iii hawthorn dichloromethane extract (0.37% plus PSE (0.24% or (iv OA/UA mixture (0.01% for 4 weeks. Compared to the control diet, hawthorn, PSE, hawthorn plus PSE and OA/UA significantly lowered plasma non-HDL (VLDL + LDL cholesterol concentrations by 8%, 9%, 21% and 6% and decreased hepatic cholesterol ester content by 9%, 23%, 46% and 22%, respectively. The cholesterol lowering effects of these ingredients were conversely associated with their capacities in increasing fecal neutral sterol excretion. In conclusion, OA and UA are responsible for the cholesterol lowering effect of hawthorn by inhibiting intestinal ACAT activity. In addition, hawthorn and particularly its bioactive compounds (OA and UA enhanced the cholesterol lowering effect of plant sterols.

  17. PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

    Science.gov (United States)

    Ohno, Yusuke; Kamiyama, Nozomi; Nakamichi, Shota; Kihara, Akio

    2017-01-01

    Lipids are the primary components of the skin permeability barrier, which is the body's most powerful defensive mechanism against pathogens. Acylceramide (ω-O-acylceramide) is a specialized lipid essential for skin barrier formation. Here, we identify PNPLA1 as the long-sought gene involved in the final step of acylceramide synthesis, esterification of ω-hydroxyceramide with linoleic acid, by cell-based assays. We show that increasing triglyceride levels by overproduction of the diacylglycerol acyltransferase DGAT2 stimulates acylceramide production, suggesting that triglyceride may act as a linoleic acid donor. Indeed, the in vitro analyses confirm that PNPLA1 catalyses acylceramide synthesis using triglyceride as a substrate. Mutant forms of PNPLA1 found in patients with ichthyosis exhibit reduced or no enzyme activity in either cell-based or in vitro assays. Altogether, our results indicate that PNPLA1 is directly involved in acylceramide synthesis as a transacylase, and provide important insights into the molecular mechanisms of skin barrier formation and of ichthyosis pathogenesis. PMID:28248318

  18. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

    Directory of Open Access Journals (Sweden)

    Brittany Ebersole

    Full Text Available We have used bioorthogonal click chemistry (BCC, a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R, a G protein-coupled receptor (GPCR crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443 of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH screen, we identified the palmitoyl acyltransferase (PAT zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  19. Engineering plant oils as high-value industrial feedstocks for biorefining - the need for underpinning cell biology research

    Energy Technology Data Exchange (ETDEWEB)

    Dyer, J.M. (US Arid-Land Agricultural Research Center, United States Dept. of Agriculture, Maricopa (US)); Mullen, R.T. (University of Guelph, Dept. of Molecular and Cellular Biology, Ontario (CA))

    2008-01-15

    Plant oils represent renewable sources of long-chain hydrocarbons that can be used as both fuel and chemical feedstocks, and genetic engineering offers an opportunity to create further high-value specialty oils for specific industrial uses. While many genes have been identified for the production of industrially important fatty acids, expression of these genes in transgenic plants has routinely resulted in a low accumulation of the desired fatty acids, indicating that significantly more knowledge of seed oil production is required before any future rational engineering designs are attempted. Here, we provide an overview of the cellular features of fatty acid desaturases, the so-called diverged desaturases, and diacylglycerol acyltransferases, three sets of enzymes that play a central role in determining the types and amounts of fatty acids that are present in seed oil, and as such, the final application and value of the oil. Recent studies of the intracellular trafficking, assembly and regulation of these enzymes have provided new insights to the mechanisms of storage oil production, and suggest that the compartmentalization of enzyme activities within specific regions or subdomains of the ER may be essential for both the synthesis of novel fatty acid structures and the channeling of these important fatty acids into seed storage oils. (au)

  20. Effects of Porphyromonas gingivalis lipopolysaccharide on the expression of key genes involved in cholesterol metabolism in macrophages

    Science.gov (United States)

    Liu, Fen; Wang, Yi; Xu, Jing; Liu, Fangqiang

    2016-01-01

    Introduction Cardiovascular diseases are positively correlated with periodontal disease. However, the molecular mechanisms linking atherosclerosis and periodontal infection are not clear. This study aimed to determine whether Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) altered the expression of genes regulating cholesterol metabolism in macrophages in the presence of low-density lipoprotein (LDL). Material and methods THP-1-derived macrophages were exposed to different concentrations (0.1, 1, 10 µg/ml) of LPS in the presence of 50 µg/ml native LDL. Macrophages were also incubated with 1 µg/ml LPS for varying times (0, 24, 48, or 72 h) in the presence of native LDL. Foam cell formation was determined by oil red O staining and cholesterol content quantification. CD36, lectin-like oxidized LDL receptor-1 (LOX-1), ATP-binding cassette G1 (ABCG1), and acetyl CoA acyltransferase 1 (ACAT1) expression levels were measured by western blot and qRT-PCR. Results Foam cell formation was induced in a time- and concentration-dependent manner as assessed by both morphological and biochemical criteria. Pg-LPS caused downregulation of CD36 and ABCG1 but upregulation of ACAT1, while LOX-1 expression was not affected (p = 0.137). Conclusions Pg-LPS appears to be an important link in the development of atherosclerosis by mechanisms targeting cholesterol homeostasis, namely, excess cholesterol ester formation via ACAT1 and reduced cellular cholesterol efflux via ABCG1. PMID:27695485

  1. Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species ( Lupinus angustifolius L. and L. mutabilis L.).

    Science.gov (United States)

    Francki, Michael G; Whitaker, Peta; Smith, Penelope M; Atkins, Craig A

    2002-11-01

    Seed triacylglycerols (TAGs) are stored as energy reserves and extracted for various end-product uses. In lupins, seed oil content varies from 16% in Lupinus mutabilisto 8% in L. angustifolius. We have shown that TAGs rapidly accumulate during mid-stages of seed development in L. mutabilis compared to the lower seed oil species, L. angustifolius. In this study, we have targeted the key enzymes of the lipid biosynthetic pathway, acetyl-CoA carboxylase (ACCase) and diacylglycerol acyltransferase (DAGAT), to determine factors regulating TAG accumulation between two lupin species. A twofold increase in ACCase activity was observed in L. mutabilis relative to L. angustifolius and correlated with rapid TAG accumulation. No difference in DAGAT activity was detected. We have identified, cloned and partially characterised a novel gene differentially expressed during TAG accumulation between L. angustifolius and L. mutabilis. The gene has some identity to the glucose dehydrogenase family previously described in barley and bacteria and the significance of its expression levels during seed development in relation to TAG accumulation is discussed. DNA sequence analysis of the promoter in both L. angustifolius and L. mutabilis identified putative matrix attachment regions and recognition sequences for transcription binding sites similar to those found in the Adh1 gene from Arabidopsis. The identical promoter regions between species indicate that differential gene expression is controlled by alternative transcription factors, accessibility to binding sites or a combination of both.

  2. Inherited and de novo deletion of the tyrosine aminotransferase gene locus at 16q22.1----q22.3 in a patient with tyrosinemia type II.

    Science.gov (United States)

    Natt, E; Westphal, E M; Toth-Fejel, S E; Magenis, R E; Buist, N R; Rettenmeier, R; Scherer, G

    1987-12-01

    Tyrosinemia II is an autosomal-recessively inherited condition caused by deficiency in the liver-specific enzyme tyrosine aminotransferase (TAT; EC 2.6.1.5). We have restudied a patient with typical symptoms of tyrosinemia II who in addition suffers from multiple congenital anomalies including severe mental retardation. Southern blot analysis using a human TAT cDNA probe revealed a complete deletion of both TAT alleles in the patient. Molecular and cytogenetic analysis of the patient and his family showed one deletion to be maternally inherited, extending over at least 27 kb and including the complete TAT structural gene, whereas loss of the second TAT allele results from a small de novo interstitial deletion, del 16 (pter----q22.1::q22.3----qter), in the paternally inherited chromosome 16. Three additional loci previously assigned to 16q22 were studied in our patient: haptoglobin (HP), lecithin: cholesterol acyltransferase (LCAT), and the metallothionein gene cluster MT1,MT2. Of these three markers, only the HP locus was found to be codeleted with the TAT locus on the del(16) chromosome.

  3. Age, sex, and lactating status regulate ghrelin secretion and GOAT mRNA levels from isolated rat stomach.

    Science.gov (United States)

    Al-Massadi, O; Crujeiras, A B; González, R C; Pardo, M; Diéguez, C; Casanueva, F F; Seoane, L M

    2010-09-01

    Ghrelin is a stomach derivate peptide involved in energy homeostasis regulation, and ghrelin O-acyltransferase (GOAT) is the enzyme responsible for ghrelin acylation. Puberty is a period characterized by profound changes in the metabolic requirements and notable variations of sexual hormone levels. On the other hand, the weaning process is a fundamental modification of the diet, which implicates several adaptations of the gastrointestinal tract physiology. Until now the direct secretion of ghrelin by the stomach in these conditions, without interferences from other organs, has never been studied. The main objective of this article was to investigate how the stomach modulates ghrelin production and secretion as well as GOAT expression on these periods of life. Gastric ghrelin secretion is regulated through postnatal life in an independent way of gastric expression and circulating levels of this hormone. The present work shows a strong regulation of gastric ghrelin secretion by estrogens. The weaning strongly regulates gastric ghrelin secretion. Animals subjected to delayed weaning present a lower body weight than the corresponding controls. For the first time, it is shown that a noticeable decrease in circulating levels of testosterone and estrogens is associated with delay of weaning. GOAT mRNA levels in the stomach are strongly regulated by age, breastfeeding, and testosterone. In conclusion, the stomach itself regulates ghrelin and GOAT production to adapt the organism to the metabolic requirements demanded through each stage of life.

  4. Altered hypothalamic protein expression in a rat model of Huntington's disease.

    Directory of Open Access Journals (Sweden)

    Wei-na Cong

    Full Text Available Huntington's disease (HD is a neurodegenerative disorder, which is characterized by progressive motor impairment and cognitive alterations. Changes in energy metabolism, neuroendocrine function, body weight, euglycemia, appetite function, and circadian rhythm can also occur. It is likely that the locus of these alterations is the hypothalamus. We used the HD transgenic (tg rat model bearing 51 CAG repeats, which exhibits similar HD symptomology as HD patients to investigate hypothalamic function. We conducted detailed hypothalamic proteome analyses and also measured circulating levels of various metabolic hormones and lipids in pre-symptomatic and symptomatic animals. Our results demonstrate that there are significant alterations in HD rat hypothalamic protein expression such as glial fibrillary acidic protein (GFAP, heat shock protein-70, the oxidative damage protein glutathione peroxidase (Gpx4, glycogen synthase1 (Gys1 and the lipid synthesis enzyme acylglycerol-3-phosphate O-acyltransferase 1 (Agpat1. In addition, there are significant alterations in various circulating metabolic hormones and lipids in pre-symptomatic animals including, insulin, leptin, triglycerides and HDL, before any motor or cognitive alterations are apparent. These early metabolic and lipid alterations are likely prodromal signs of hypothalamic dysfunction. Gaining a greater understanding of the hypothalamic and metabolic alterations that occur in HD, could lead to the development of novel therapeutics for early interventional treatment of HD.

  5. Downregulation of miR-150 Expression by DNA Hypermethylation Is Associated with High 2-Hydroxy-(4-methylthio)butanoic Acid-Induced Hepatic Cholesterol Accumulation in Nursery Piglets.

    Science.gov (United States)

    Jia, Yimin; Ling, Mingfa; Zhang, Luchu; Jiang, Shuxia; Sha, Yusheng; Zhao, Ruqian

    2016-10-12

    Excess 2-hydroxy-(4-methylthio)butanoic acid (HMB) supplementation induces hyperhomocysteinemia, which contributes to hepatic cholesterol accumulation. However, it is unclear whether and how high levels of HMB break hepatic cholesterol homeostasis in nursery piglets. In this study, HMB oversupplementation suppressed food intake and decreased body weight in nursery piglets. Hyperhomocysteinemia and higher hepatic cholesterol accumulation were observed in HMB groups. Accordingly, HMB significantly increased the protein content of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and glycine N-methyltransferase (GNMT) but decreased that of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1). Significant downregulation of miR-150, miR-181d-5p, and miR-296-3p targeting the 3'-untranslated regions (UTRs) of GNMT and HMGCR was detected in the liver of HMB-treated piglets, and their functional validation was confirmed by dual-luciferase reporter assay. Furthermore, hypermethylation of miR-150 promoter was detected in association with suppressed miR-150 expression in the livers of HMB-treated piglets. This study indicated a new mechanism of hepatic cholesterol unhomeostasis by dietary methyl donor supplementation.

  6. Effect of chlorpromazine on lipid metabolism in aortas from cholesterol-fed rabbits and normal rats, in vitro: inhibition of sterol esterification and modification of phospholipid synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Bell, F.P.

    1983-06-01

    Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes and in intact arterial tissue from the rat and cholesterol-fed rabbit in vitro. In isolated rabbit arterial microsomes, CPZ resulted in a concentration-dependent inhibition of ACAT with 50% inhibition of (1-14C)oleoylCoA incorporation into (14C)cholesteryl esters occurring at 0.1 mM CPZ. CPZ also effectively inhibited the incorporation of (14C)oleate into triglycerides without affecting incorporation into diglycerides. Additionally, CPZ altered the pattern of arterial phospholipids synthesized from (1-14C)oleate. Incorporation into phosphatidylcholine was depressed while incorporation into phosphatidylinositol was increased. Since diglyceride synthesis appeared to be unaffected by CPZ, a redirection of phosphatidic acid into the CDP-diglyceride pathway of glycerolipid synthesis does not adequately account for the effect of CPZ on arterial phospholipid and triglyceride synthesis in these experiments.

  7. Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Wang, Xi; Xiong, Xiaochao; Sa, Na; Roje, Sanja; Chen, Shulin

    2016-07-01

    With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

  8. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Directory of Open Access Journals (Sweden)

    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  9. Massive bowel resection upregulates the intestinal mRNA expression levels of cellular retinol-binding protein II and apolipoprotein A-IV and alters the intestinal vitamin A status in rats.

    Science.gov (United States)

    Hebiguchi, Taku; Mezaki, Yoshihiro; Morii, Mayako; Watanabe, Ryo; Yoshikawa, Kiwamu; Miura, Mitsutaka; Imai, Katsuyuki; Senoo, Haruki; Yoshino, Hiroaki

    2015-03-01

    Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.

  10. Retinol status and expression of retinol-related proteins in methionine-choline deficient rats.

    Science.gov (United States)

    Miyazaki, Hiroshi; Takitani, Kimitaka; Koh, Maki; Inoue, Akiko; Kishi, Kanta; Tamai, Hiroshi

    2014-01-01

    Retinol and its derivative, retinoic acid, have pleiotropic functions including vision, immunity, hematopoiesis, reproduction, cell differentiation/growth, and development. Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases in developed countries and encompasses a broad spectrum of forms, ranging from steatosis to steatohepatitis, which develops further to cirrhosis. Retinol status has an important role in liver homeostasis. The purpose of this study was to evaluate the retinol status and expression of retinol-related proteins, including enzymes and binding proteins, in methionine-choline deficient (MCD) rats as a model of NAFLD. We examined retinol levels in the plasma and liver and gene expression for β-carotene 15,15'-monooxygenase (BCMO), lecithIn: retinol acyltransferase (LRAT), aldehyde dehydrogenase 1A1 (ALDH1A1), ALDH1A2, and cellular retinol binding protein (CRBP)-I in MCD rats. The plasma retinol levels in MCD rats were lower than those in the controls, whereas hepatic retinol levels in MCD rats were higher. BCMO expression in the intestine and liver in MCD rats was lower, whereas that in the testes and the kidneys was higher than in control rats. Expression of LRAT, CRBP-I, ALDH1A1, and ALDH1A2 in the liver of MCD rats was also higher. Altered expression of retinol-related proteins may affect retinol status in NAFLD.

  11. Casein phosphopeptides and CaCl2 increase penicillin production and cause an increment in microbody/peroxisome proteins in Penicillium chrysogenum.

    Science.gov (United States)

    Domínguez-Santos, Rebeca; Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Ibáñez, Ana; Morales, Alejandro; Martín, Juan-Francisco

    2017-03-06

    Transport of penicillin intermediates and penicillin secretion are still poorly characterized in Penicillium chrysogenum (re-identified as Penicillium rubens). Calcium (Ca(2+)) plays an important role in the metabolism of filamentous fungi, and casein phosphopeptides (CPP) are involved in Ca(2+) internalization. In this study we observe that the effect of CaCl2 and CPP is additive and promotes an increase in penicillin production of up to 10-12 fold. Combination of CaCl2 and CPP greatly promotes expression of the three penicillin biosynthetic genes. Comparative proteomic analysis by 2D-DIGE, identified 39 proteins differentially represented in P. chrysogenum Wisconsin 54-1255 after CPP/CaCl2 addition. The most interesting group of overrepresented proteins were a peroxisomal catalase, three proteins of the methylcitrate cycle, two aminotransferases and cystationine β-synthase, which are directly or indirectly related to the formation of penicillin amino acid precursors. Importantly, two of the enzymes of the penicillin pathway (isopenicillin N synthase and isopenicillin N acyltransferase) are clearly induced after CPP/CaCl2 addition. Most of these overrepresented proteins are either authentic peroxisomal proteins or microbody-associated proteins. This evidence suggests that addition of CPP/CaCl2 promotes the formation of penicillin precursors and the penicillin biosynthetic enzymes in peroxisomes and vesicles, which may be involved in transport and secretion of penicillin.

  12. Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells.

    Science.gov (United States)

    Kiel, Jan A K W; van den Berg, Marco A; Fusetti, Fabrizia; Poolman, Bert; Bovenberg, Roel A L; Veenhuis, Marten; van der Klei, Ida J

    2009-05-01

    In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.

  13. Mammalian N-acetylglutamate synthase.

    Science.gov (United States)

    Morizono, Hiroki; Caldovic, Ljubica; Shi, Dashuang; Tuchman, Mendel

    2004-04-01

    N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

  14. The biosynthesis of hydroxycinnamoyl quinate esters and their role in the storage of cocaine in Erythroxylum coca.

    Science.gov (United States)

    Torre, José Carlos Pardo; Schmidt, Gregor W; Paetz, Christian; Reichelt, Michael; Schneider, Bernd; Gershenzon, Jonathan; D'Auria, John C

    2013-07-01

    Complexation of alkaloids is an important strategy plants utilize to facilitate storage in vacuoles and avoid autotoxicity. Previous studies have implicated hydroxycinnamoyl quinate esters in the complexation of purine alkaloids in Coffea arabica. The goal of this study was to determine if Erythroxylum coca uses similar complexation agents to store abundant tropane alkaloids, such as cocaine and cinnamoyl cocaine. Metabolite analysis of various E. coca organs established a close correlation between levels of coca alkaloids and those of two hydroxycinnamoyl esters of quinic acid, chlorogenic acid and 4-coumaroyl quinate. The BAHD acyltransferase catalyzing the final step in hydroxycinnamoyl quinate biosynthesis was isolated and characterized, and its gene expression found to correlate with tropane alkaloid accumulation. A physical interaction between chlorogenic acid and cocaine was observed and quantified in vitro using UV and NMR spectroscopic methods yielding similar values to those reported for a caffeine chlorogenate complex in C. arabica. These results suggest that storage of cocaine and other coca alkaloids in large quantities in E. coca involves hydroxycinnamoyl quinate esters as complexation partners.

  15. A metabolomics study of the inhibitory effect of 17-beta-estradiol on osteoclast proliferation and differentiation.

    Science.gov (United States)

    Liu, Xiaoyan; Liu, Yanqiu; Cheng, Mengchun; Zhang, Xiaozhe; Xiao, Hongbin

    2015-02-01

    Estradiol is a major drug used clinically to alleviate osteoporosis, partly through inhibition of the activity of osteoclasts, which play a crucial role in bone resorption. So far, little is known about the effects of estradiol on osteoclast metabolism. In this study, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC/MS)-based metabolomics strategy was used to investigate the metabolite response to 17β-estradiol in mouse osteoclast RAW264.7, a commonly used cell model for studying osteoporosis. Our results showed that the application of estradiol altered the levels of 27 intracellular metabolites, including lysophosphatidylcholines (LysoPCs), other lipids and amino acid derivants. The changes of all the 27 metabolites were observed in the study of estradiol induced osteoclast proliferation inhibition (1 μM estradiol applied), while the changes of only 18 metabolites were observed in the study of differentiation inhibition (0.1 μM estradiol applied). Further pathway impact analysis determined glycerophospholipid metabolism as the main potential target pathway of estradiol, which was further confirmed by LCAT (phosphatidylcholine-sterol acyltransferase) activity changes and lipid peroxidative product (MDA, methane dicarboxylic aldehyde) changes caused by estradiol. Additionally, we found that estradiol significantly decreased intracellular oxidative stress during cell proliferation but not during cell differentiation. Our study suggested that estradiol generated a highly condition-dependent influence on osteoclast metabolism.

  16. Molecular Dynamics Simulation and Experimental Studies of Gold Nanoparticle Templated HDL-like Nanoparticles for Cholesterol Metabolism Therapeutics.

    Science.gov (United States)

    Lai, Cheng-Tsung; Sun, Wangqiang; Palekar, Rohun U; Thaxton, C Shad; Schatz, George C

    2017-01-18

    High-density lipoprotein (HDL) plays an important role in the transport and metabolism of cholesterol. Mimics of HDL are being explored as potentially powerful therapeutic agents for removing excess cholesterol from arterial plaques. Gold nanoparticles (AuNPs) functionalized with apolipoprotein A-I and with the lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] have been demonstrated to be robust acceptors of cellular cholesterol. However, detailed structural information about this functionalized HDL AuNP is still lacking. In this study, we have used X-ray photoelectron spectroscopy and lecithin/cholesterol acyltransferase activation experiments together with coarse-grained and all-atom molecular dynamics simulations to model the structure and cholesterol uptake properties of the HDL AuNP construct. By simulating different apolipoprotein-loaded AuNPs, we find that lipids are oriented differently in regions with and without apoA-I. We also show that in this functionalized HDL AuNP, the distribution of cholesteryl ester maintains a reverse concentration gradient that is similar to the gradient found in native HDL.

  17. Determination of internal controls for quantitative gene expression of Isochrysis zhangjiangensis at nitrogen stress condition

    Science.gov (United States)

    Wu, Shuang; Zhou, Jiannan; Cao, Xupeng; Xue, Song

    2016-02-01

    Isochrysis zhangjiangensis is a potential marine microalga for biodiesel production, which accumulates lipid under nitrogen limitation conditions, but the mechanism on molecular level is veiled. Quantitative real-time polymerase chain reaction (qPCR) provides the possibility to investigate the gene expression levels, and a valid reference for data normalization is an essential prerequisite for firing up the analysis. In this study, five housekeeping genes, actin (ACT), α-tubulin (TUA), ß-tubulin (TUB), ubiquitin (UBI), 18S rRNA (18S) and one target gene, diacylglycerol acyltransferase (DGAT), were used for determining the reference. By analyzing the stabilities based on calculation of the stability index and on operating the two types of software, geNorm and bestkeeper, it showed that the reference genes widely used in higher plant and microalgae, such as UBI, TUA and 18S, were not the most stable ones in nitrogen-stressed I. zhangjiangensis, and thus are not suitable for exploring the mRNA expression levels under these experimental conditions. Our results show that ACT together with TUB is the most feasible internal control for investigating gene expression under nitrogen-stressed conditions. Our findings will contribute not only to future qPCR studies of I. zhangjiangensis, but also to verification of comparative transcriptomics studies of the microalgae under similar conditions.

  18. Assessment of single nucleotide polymorphisms in genes residing on chromosomes 14 and 29 for association with carcass composition traits in Bos indicus cattle.

    Science.gov (United States)

    Casas, E; White, S N; Riley, D G; Smith, T P L; Brenneman, R A; Olson, T A; Johnson, D D; Coleman, S W; Bennett, G L; Chase, C C

    2005-01-01

    Objective of this study was to assess the association of SNP in the diacylglycerol O-acyltransferase 1 (DGAT1), thyroglobulin (TG), and micromolar calcium-activated neutral protease (CAPN1) genes with carcass composition and meat quality traits in Bos indicus cattle. A population of Brahman calves (n = 479) was developed in central Florida from 1996 to 2000. Traits analyzed were ADG, hip height, slaughter weight, fat thickness, HCW, marbling score, LM area, estimated KPH fat, yield grade, retail yield, sensory panel tenderness score, carcass hump height, and cooked meat tenderness measured as Warner-Bratzler shear force at 7, 14, and 21 d postmortem. Single nucleotide polymorphisms previously reported in the TG and DGAT1 genes were used as markers on chromosome 14. Two previously reported and two new SNP in the CAPN1 gene were used as markers on chromosome 29. One SNP in CAPN1 was uninformative, and another one was associated with tenderness score (P Brahman population than in reported allele frequencies in Bos taurus populations. The results suggest that the use of molecular marker information developed in Bos taurus populations to Bos indicus populations may require development of appropriate additional markers.

  19. Deficient Cholesterol Esterification in Plasma of apoc2 Knockout Zebrafish and Familial Chylomicronemia Patients

    Science.gov (United States)

    Liu, Chao; Gaudet, Daniel; Miller, Yury I.

    2017-01-01

    Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Apolipoprotein C-II (APOC2) is an obligatory cofactor for lipoprotein lipase (LPL), the major enzyme catalyzing plasma triglyceride hydrolysis. We have created an apoc2 knockout zebrafish model, which mimics the familial chylomicronemia syndrome (FCS) in human patients with a defect in the APOC2 or LPL gene. In this study, we measured plasma levels of free cholesterol (FC) and cholesterol esters (CE) and found that apoc2 mutant zebrafish have a significantly higher FC to CE ratio (FC/CE), when compared to the wild type. Feeding apoc2 mutant zebrafish a low-fat diet reduced triglyceride levels but not the FC/CE ratio. In situ hybridization and qPCR results demonstrated that the hepatic expression of lecithin-cholesterol acyltransferase (lcat), the enzyme responsible for esterifying plasma FC to CE, and of apolipoprotein A-I, a major protein component of HDL, were dramatically decreased in apoc2 mutants. Furthermore, the FC/CE ratio was significantly increased in the whole plasma and in a chylomicron-depleted fraction of human FCS patients. The FCS plasma LCAT activity was significantly lower than that of healthy controls. In summary, this study, using a zebrafish model and human patient samples, reports for the first time the defect in plasma cholesterol esterification associated with LPL deficiency. PMID:28107429

  20. Moro orange juice prevents fatty liver in mice

    Institute of Scientific and Technical Information of China (English)

    Federico Salamone; Marco Giorgio; Fabio Galvano; Giovanni Li Volti; Lucilla Titta; Lidia Puzzo; Ignazio Barbagallo; Francesco La Delia; Shira Zelber-Sagi; Michele Malaguarnera; Pier Giuseppe Pelicci

    2012-01-01

    AIM:To establish if the juice of Moro,an anthocyaninrich orange,may improve liver damage in mice with diet-induced obesity.METHODS:Eight-week-old mice were fed a high-fat diet (HFD) and were administrated water or Moro juice for 12 wk.Liver morphology,gene expression of lipid transcription factors,and metabolic enzymes were assessed.RESULTS:Mice fed HFD displayed increased body weight,insulin resistance and dyslipidemia.Moro juice administration limited body weight gain,enhanced insulin sensitivity,and decreased serum triglycerides and total cholesterol.Mice fed HFD showed liver steatosis associated with ballooning.Dietary Moro juice markedly improved liver steatosis by inducing the expression of peroxisome proliferator-activated receptor-a and its target gene acylCoA-oxidase,a key enzyme of lipid oxidation.Consistently,Moro juice consumption suppressed the expression of liver X receptor-o and its target gene fatty acid synthase,and restored liver glycerol-3-phosphate acyltransferase 1 activity.CONCLUSION:Moro juice counteracts liver steatogenesis in mice with diet-induced obesity and thus may represent a promising dietary option for the prevention of fatty liver.