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Sample records for acyltransferases

  1. Glycerophosphate/Acylglycerophosphate Acyltransferases

    Directory of Open Access Journals (Sweden)

    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. 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

  3. Acyl-coenzyme A:cholesterol acyltransferases

    OpenAIRE

    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...

  4. 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.

  5. Lecithin : cholesterol acyltransferase: old friend or foe in atherosclerosis?

    NARCIS (Netherlands)

    Kunnen, S.; Eck, van M.

    2012-01-01

    Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme that catalyzes the esterification of free cholesterol in plasma lipoproteins and plays a critical role in high-density lipoprotein (HDL) metabolism. Deficiency leads to accumulation of nascent preβ-HDL due to impaired maturation of HDL

  6. Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII

    DEFF Research Database (Denmark)

    Musiol-Kroll, Ewa Maria; Zubeil, Florian; Schafhauser, Thomas

    2017-01-01

    During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmal...... nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by "click" chemistry....

  7. Cellular Pregnenolone Esterification by Acyl-CoA:Cholesterol Acyltransferase*

    Science.gov (United States)

    Rogers, Maximillian A.; Liu, Jay; Kushnir, Mark M.; Bryleva, Elena; Rockwood, Alan L.; Meikle, A. Wayne; Shapiro, David; Vaisman, Boris L.; Remaley, Alan T.; Chang, Catherine C. Y.; Chang, Ta-Yuan

    2012-01-01

    Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the Vmax for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30–50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues. PMID:22474282

  8. Polyketide Proofreading by an Acyltransferase-like Enzyme

    Science.gov (United States)

    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

    2012-01-01

    SUMMARY 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. PMID:22444588

  9. The Uncommon Enzymology of Cis-Acyltransferase Assembly Lines.

    Science.gov (United States)

    Keatinge-Clay, Adrian T

    2017-04-26

    The enzymology of 135 assembly lines containing primarily cis-acyltransferase modules is comprehensively analyzed, with greater attention paid to less common phenomena. Diverse online transformations, in which the substrate and/or product of the reaction is an acyl chain bound to an acyl carrier protein, are classified so that unusual reactions can be compared and underlying assembly-line logic can emerge. As a complement to the chemistry surrounding the loading, extension, and offloading of assembly lines that construct primarily polyketide products, structural aspects of the assembly-line machinery itself are considered. This review of assembly-line phenomena, covering the literature up to 2017, should thus be informative to the modular polyketide synthase novice and expert alike.

  10. 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.

    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

  11. In Vivo and in Vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase*

    Science.gov (United States)

    Pan, Xue; Chen, Guanqun; Kazachkov, Michael; Greer, Michael S.; Caldo, Kristian Mark P.; Zou, Jitao; Weselake, Randall J.

    2015-01-01

    Seed oils of flax (Linum usitatissimum L.) and many other plant species contain substantial amounts of polyunsaturated fatty acids (PUFAs). Phosphatidylcholine (PC) is the major site for PUFA synthesis. The exact mechanisms of how these PUFAs are channeled from PC into triacylglycerol (TAG) needs to be further explored. By using in vivo and in vitro approaches, we demonstrated that the PC deacylation reaction catalyzed by the reverse action of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) can transfer PUFAs on PC directly into the acyl-CoA pool, making these PUFAs available for the diacylglycerol acyltransferase (DGAT)-catalyzed reaction for TAG production. Two types of yeast mutants were generated for in vivo and in vitro experiments, respectively. Both mutants provide a null background with no endogenous TAG forming capacity and an extremely low LPCAT activity. In vivo experiments showed that co-expressing flax DGAT1-1 and LPCAT1 in the yeast quintuple mutant significantly increased 18-carbon PUFAs in TAG with a concomitant decrease of 18-carbon PUFAs in phospholipid. We further showed that after incubation of sn-2-[14C]acyl-PC, formation of [14C]TAG was only possible with yeast microsomes containing both LPCAT1 and DGAT1-1. Moreover, the specific activity of overall LPCAT1 and DGAT1-1 coupling process exhibited a preference for transferring 14C-labeled linoleoyl or linolenoyl than oleoyl moieties from the sn-2 position of PC to TAG. Together, our data support the hypothesis of biochemical coupling of the LPCAT1-catalyzed reverse reaction with the DGAT1-1-catalyzed reaction for incorporating PUFAs into TAG. This process represents a potential route for enriching TAG in PUFA content during seed development in flax. PMID:26055703

  12. Lecithin:cholesterol acyltransferase: old friend or foe in atherosclerosis?

    Science.gov (United States)

    Kunnen, Sandra; Van Eck, Miranda

    2012-01-01

    Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme that catalyzes the esterification of free cholesterol in plasma lipoproteins and plays a critical role in high-density lipoprotein (HDL) metabolism. Deficiency leads to accumulation of nascent preβ-HDL due to impaired maturation of HDL particles, whereas enhanced expression is associated with the formation of large, apoE-rich HDL1 particles. In addition to its function in HDL metabolism, LCAT was believed to be an important driving force behind macrophage reverse cholesterol transport (RCT) and, therefore, has been a subject of great interest in cardiovascular research since its discovery in 1962. Although half a century has passed, the importance of LCAT for atheroprotection is still under intense debate. This review provides a comprehensive overview of the insights that have been gained in the past 50 years on the biochemistry of LCAT, the role of LCAT in lipoprotein metabolism and the pathogenesis of atherosclerosis in animal models, and its impact on cardiovascular disease in humans. PMID:22566575

  13. Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2.

    Science.gov (United States)

    Tarui, Megumi; Shindou, Hideo; Kumagai, Kazuo; Morimoto, Ryo; Harayama, Takeshi; Hashidate, Tomomi; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Nagase, Takahide; Shimizu, Takao

    2014-07-01

    Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

    Directory of Open Access Journals (Sweden)

    Michelle A Blaskovich

    Full Text Available Lysophosphatidic acid acyltransferase (LPAAT-β is a phosphatidic acid (PA generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta

    NARCIS (Netherlands)

    Ofman, R.; Wanders, R. J.

    1994-01-01

    The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to

  17. Solubilization of the plastidial lysophosphatidylcholine acyltransferase from Allium porrum leaves: towards plants devoid of eukaryotic plastid lipids?

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    Akermoun, M; Testet, E; Cassagne, C; Bessoule, J J

    2000-12-01

    To analyse the involvement of the plastidial lysophosphatidylcholine (lyso-PC) acyltransferase in the import of the extraplastidial lipid precursors required for eukaryotic plastid lipid synthesis, we plan to obtain transgenic plants. Since no sequence of lyso-PC acyltransferase is known, the purification of this enzyme has been undertaken to establish its sequence. First we determined the conditions allowing the solubilization of this membrane-bound enzyme. It is shown that by using CHAPS as a detergent, a lyso-PC acyltransferase activity is associated with the solubilized proteins.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Compared with Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol.

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    Temel, Ryan E; Gebre, Abraham K; Parks, John S; Rudel, Lawrence L

    2003-11-28

    The capacity of acyl-CoA:cholesterol O-acyltransferase (ACAT) 2 to differentiate cholesterol from the plant sterol, sitosterol, was compared with that of the sterol esterifying enzymes, ACAT1 and lecithin:cholesterol acyltransferase (LCAT). Cholesterol-loaded microsomes from transfected cells containing either ACAT1 or ACAT2 exhibited significantly more ACAT activity than their sitosterol-loaded counterparts. In sitosterol-loaded microsomes, both ACAT1 and ACAT2 were able to esterify sitosterol albeit with lower efficiencies than cholesterol. The mass ratios of cholesterol ester to sitosterol ester formed by ACAT1 and ACAT2 were 1.6 and 7.2, respectively. Compared with ACAT1, ACAT2 selectively esterified cholesterol even when sitosterol was loaded into the microsomes. To further characterize the difference in sterol specificity, ACAT1 and ACAT2 were compared in intact cells loaded with either cholesterol or sitosterol. Despite a lower level of ACAT activity, the ACAT1-expressing cells esterified 4-fold more sitosterol than the ACAT2 cells. The data showed that compared with ACAT1, ACAT2 displayed significantly greater selectively for cholesterol compared with sitosterol. The plasma cholesterol esterification enzyme lecithin:cholesterol acyltransferase was also compared. With recombinant high density lipoprotein particles, the esterification rate of cholesterol by LCAT was only 15% greater than for sitosterol. Thus, LCAT was able to efficiently esterify both cholesterol and sitosterol. In contrast, ACAT2 demonstrated a strong preference for cholesterol rather than sitosterol. This sterol selectivity by ACAT2 may reflect a role in the sorting of dietary sterols during their absorption by the intestine in vivo.

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

    OpenAIRE

    Netherland, Courtney; Thewke, Douglas P.

    2010-01-01

    Acyl-coenzymeA:cholesterol acyltransferase (ACAT) catalyzes the intracellular synthesis of cholesteryl esters (CE). Both ACAT isoforms, ACAT1 and ACAT2, play key roles in the pathophysiology of atherosclerosis and ACAT inhibition retards atherosclerosis in animal models. Rimonabant, a type 1 cannabinoid receptor (CB1) antagonist, produces anti-atherosclerotic effects in humans and animals by mechanisms which are not completely understood. Rimonabant is structurally similar to two other cannab...

  2. Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1

    OpenAIRE

    Chen, Hubert C.; Smith, Steven J.; Ladha, Zuleika; Jensen, Dalan R.; Ferreira, Luis D.; Pulawa, Leslie K.; McGuire, James G.; Pitas, Robert E.; Robert H Eckel; Farese, Robert V.

    2002-01-01

    Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in mammalian triglyceride synthesis. DGAT1-deficient mice are resistant to diet-induced obesity through a mechanism involving increased energy expenditure. Here we show that these mice have decreased levels of tissue triglycerides, as well as increased sensitivity to insulin and to leptin. Importantly, DGAT1 deficiency protects against insulin resistance and obesity in agouti...

  3. Overexpression of diacylglycerol acyltransferase in Yarrowia lipolytica affects lipid body size, number and distribution.

    Science.gov (United States)

    Gajdoš, Peter; Ledesma-Amaro, Rodrigo; Nicaud, Jean-Marc; Čertík, Milan; Rossignol, Tristan

    2016-09-01

    In the oleaginous yeast Yarrowia lipolytica, the diacylglycerol acyltransferases (DGATs) are major factors for triacylglycerol (TAG) synthesis. The Q4 strain, in which the four acyltransferases have been deleted, is unable to accumulate lipids and to form lipid bodies (LBs). However, the expression of a single acyltransferase in this strain restores TAG accumulation and LB formation. Using this system, it becomes possible to characterize the activity and specificity of an individual DGAT. Here, we examined the effects of DGAT overexpression on lipid accumulation and LB formation in Y. lipolytica Specifically, we evaluated the consequences of introducing one or two copies of the Y. lipolytica DGAT genes YlDGA1 and YlDGA2 Overall, multi-copy DGAT overexpression increased the lipid content of yeast cells. However, the size and distribution of LBs depended on the specific DGAT overexpressed. YlDGA2 overexpression caused the formation of large LBs, while YlDGA1 overexpression generated smaller but more numerous LBs. This phenotype was accentuated through the addition of a second copy of the overexpressed gene and might be linked to the distinct subcellular localization of each DGAT, i.e. YlDga1 being localized in LBs, while YlDga2 being localized in a structure strongly resembling the endoplasmic reticulum. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

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    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.

  5. 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

    and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane...

  6. Acyl-coenzyme A:cholesterol acyltransferase inhibition ameliorates proteinuria, hyperlipidemia, lecithin-cholesterol acyltransferase, SRB-1, and low-denisty lipoprotein receptor deficiencies in nephrotic syndrome.

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    Vaziri, N D; Liang, K H

    2004-07-27

    Nephrotic syndrome (NS) is associated with hyperlipidemia, altered lipid regulatory enzymes and receptors, and increased risk of progressive renal and cardiovascular diseases. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes intracellular esterification of cholesterol and plays an important role in production of apolipoprotein B-containing lipoproteins, regulation of cholesterol-responsive proteins, and formation of foam cells. Because hepatic ACAT-2 is markedly upregulated in NS, we tested the hypothesis that inhibition of ACAT may improve cholesterol metabolism in NS. Rats with puromycin-induced NS were treated with either the ACAT inhibitor CI-976 or placebo for 2 weeks. Normal rats served as controls. Plasma lipids, renal function, and key lipid regulatory factors were measured. Untreated NS rats showed heavy proteinuria; hypoalbuminemia; elevated plasma cholesterol, triglyceride, LDL, VLDL, and total cholesterol-to-HDL cholesterol ratio; increased hepatic ACAT activity, ACAT-2 mRNA, and ACAT-2 protein; and reduced LDL receptor, HDL receptor, otherwise known as scavenger receptor B-1 (SRB-1) and plasma lecithin-cholesterol acyltransferase (LCAT). ACAT inhibitor reduced plasma cholesterol and triglycerides, normalized total cholesterol-to-HDL cholesterol ratio, and lowered hepatic ACAT activity without changing ACAT-2 mRNA or protein. This was accompanied by near normalizations of plasma LCAT, hepatic SRB-1, and LDL receptor and a significant amelioration of proteinuria and hypoalbuminemia. Pharmacological inhibition of ACAT reverses NS-induced LDL receptor, HDL receptor, and LCAT deficiencies; improves plasma lipid profile; and ameliorates proteinuria in nephrotic animals. Further studies are needed to explore the effect of ACAT inhibition in nephrotic humans.

  7. Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape.

    OpenAIRE

    Little, D.; Weselake, R; Pomeroy, K; Furukawa-Stoffer, T; Bagu, J

    1994-01-01

    Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity...

  8. A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis.

    Science.gov (United States)

    Rani, Sapa Hima; Saha, Saikat; Rajasekharan, Ram

    2013-01-01

    The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

  9. Overexpression of Glycerol-3-Phosphate Acyltransferase from Suaeda salsa Improves Salt Tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Na Sui

    2017-08-01

    Full Text Available Glycerol-3-phosphate acyltransferase is the first acyl esterifying enzyme in phosphatidylglycerol (PG synthesis process. In this study, we isolated and characterized the glycerol-3-phosphate acyltransferase (GPAT gene from Suaeda salsa (S. salsa and obtained the full length of the GPAT gene from S. salsa (SsGPAT by 5′ and 3′ RACE. The clone contained an open reading frame (ORF of 1167 bp nucleotides that comprised of 388 amino acid residues. Real-time PCR revealed that the mRNA accumulation of GPAT in S. salsa was induced by salt stress. The highest expression levels were observed when S. salsa leaves were exposed to 300 mM NaCl treatment. At the germination stage, the germination rate and root length of overexpressed Arabidopsis strains were significantly higher than WT under different concentrations of NaCl treatments, while the inhibitory effect was significantly severe in T-DNA insertion mutant strains. In the seedling stage, chlorophyll content, the photochemical efficiency of PSII, PSI oxidoreductive activity (ΔI/Io, and the unsaturated fatty acid content of PG decreased less in overexpressed strains and more in mutant strains than that in WT under salt stress. These results suggest that the overexpression of SsGPAT in Arabidopsis enhances salt tolerance and alleviates the photoinhibition of PSII and PSI under salt stress by improving the unsaturated fatty acid content of PG.

  10. Mouse ghrelin-O-acyltransferase (GOAT) plays a critical role in bile acid reabsorption.

    Science.gov (United States)

    Kang, Kihwa; Schmahl, Jennifer; Lee, Jong-Min; Garcia, Karen; Patil, Ketan; Chen, Amelia; Keene, Michelle; Murphy, Andrew; Sleeman, Mark W

    2012-01-01

    Ghrelin is a unique peptide gut hormone that requires post-translational modification to stimulate both feeding and growth hormone release. Ghrelin O-acyltransferase (GOAT) was identified as a specific acyl-transferase for ghrelin, and recent genetic deletion studies of the Goat gene (Goat(-/-)) uncovered the role of ghrelin in the regulation of glucose homeostasis. To further understand the physiological functions of the GOAT/ghrelin system, we have conducted a metabolomic and microarray profile of Goat-null mice, as well as determined Goat expression in different tissues using the lacZ reporter gene. Serum metabolite profile analysis revealed that Goat(-/-) mice exhibited increased secondary bile acids >2.5-fold. This was attributed to increased mRNA and protein expression of the ileal sodium-dependent bile acid transporter (ISBT) in the intestinal and biliary tract. Increased expression of additional solute carrier proteins, including Slc5a12 (>10-fold) were also detected in the small intestine and bile duct. Goat staining was consistently observed in the pituitary glands, stomach, and intestines, and to a lesser extent in the gallbladder and pancreatic duct. This is the first report that the GOAT/ghrelin system regulates bile acid metabolism, and these findings suggest a novel function of GOAT in the regulation of intestinal bile acid reabsorption..

  11. 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

  12. Plasma lecithin : cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men

    NARCIS (Netherlands)

    Dullaart, R. P. F.; Perton, F.; Kappelle, P.J.W.H.; de Vries, R.; Sluiter, W. J.; van Tol, A.

    Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL We determined the relationship of plasma high-sensitivity

  13. Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana.

    Directory of Open Access Journals (Sweden)

    Donghui Zhang

    Full Text Available In the remodeling pathway for the synthesis of phosphatidylcholine (PC, acyl-CoA-dependent lysophosphatidylcholine (lysoPC acyltransferase (LPCAT catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2. Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△ disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA and α-linolenic acid (18:3n3, ALA into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3, while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid

  14. A Cytosolic Acyltransferase Contributes to Triacylglycerol Synthesis in Sucrose-Rescued Arabidopsis Seed Oil Catabolism Mutants1[W][OA

    Science.gov (United States)

    Hernández, M. Luisa; Whitehead, Lynne; He, Zhesi; Gazda, Valeria; Gilday, Alison; Kozhevnikova, Ekaterina; Vaistij, Fabián E.; Larson, Tony R.; Graham, Ian A.

    2012-01-01

    Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the β-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase. PMID:22760209

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

    Energy Technology Data Exchange (ETDEWEB)

    Eudes, Aymerick [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Mouille, Maxence [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Robinson, David S. [Joint BioEnergy Institute, Emeryville, CA (United States); Benites, Veronica T. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); San Francisco State Univ., San Francisco, CA (United States); Wang, George [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Roux, Lucien [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Ecole Polytechnique Federale de Lausanne, Lausanne (Switzerland); Tsai, Yi-Lin [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Baidoo, Edward E. K. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Chiu, Tsan-Yu [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Heazlewood, Joshua L. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); The Univ. of Melbourne, Melbourne, VIC (Australia); Scheller, Henrik V. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Mukhopadhyay, Aindrila [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Keasling, Jay D. [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark); Deutsch, Samuel [Joint BioEnergy Institute, Emeryville, CA (United States); Loqué, Dominique [Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. Claude Bernard Lyon 1, Villeurbanne (France)

    2016-11-21

    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 of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical 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. As a result, for the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters

  16. 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.

  17. First identification of xanthone sulfonamides as potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors.

    Science.gov (United States)

    Hu, Honggang; Liao, Hongli; Zhang, Jun; Wu, Weifeng; Yan, Jufang; Yan, Yonghong; Zhao, Qingjie; Zou, Yan; Chai, Xiaoyun; Yu, Shichong; Wu, Qiuye

    2010-05-15

    Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) would be useful anti-atherogenic agents, since an absence of ACAT affects the absorption and transformation of cholesterol, indirectly resulting in the reduction of cholesteryl ester accumulation in blood vessels. This report discloses xanthone sulfonamides as novel class small molecule inhibitors of ACAT. A series of xanthone sulfonamides were synthesized and evaluated to result in the identification of several potent ACAT inhibitors, among which 2n proved to be more potent than the positive control Sandoz58-35. Moreover, a molecular model for the binding between 2n and the active site of ACAT-2 was provided based computational docking results. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Increased insulin and leptin sensitivity in mice lacking acyl CoA:diacylglycerol acyltransferase 1.

    Science.gov (United States)

    Chen, Hubert C; Smith, Steven J; Ladha, Zuleika; Jensen, Dalan R; Ferreira, Luis D; Pulawa, Leslie K; McGuire, James G; Pitas, Robert E; Eckel, Robert H; Farese, Robert V

    2002-04-01

    Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final step in mammalian triglyceride synthesis. DGAT1-deficient mice are resistant to diet-induced obesity through a mechanism involving increased energy expenditure. Here we show that these mice have decreased levels of tissue triglycerides, as well as increased sensitivity to insulin and to leptin. Importantly, DGAT1 deficiency protects against insulin resistance and obesity in agouti yellow mice, a model of severe leptin resistance. In contrast, DGAT1 deficiency did not affect energy and glucose metabolism in leptin-deficient (ob/ob) mice, possibly due in part to a compensatory upregulation of DGAT2 expression in the absence of leptin. Our results suggest that inhibition of DGAT1 may be useful in treating insulin resistance and leptin resistance in human obesity.

  19. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase.

    Science.gov (United States)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A; Tesmer, John J G

    2015-03-02

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  20. Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase.

    Science.gov (United States)

    Reshetnyak, Yana; Tchedre, Kissaou T; Nair, Maya P; Pritchard, P Haydn; Lacko, Andras G

    2006-08-01

    Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).

  1. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    Science.gov (United States)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A.; Tesmer, John J. G.

    2015-03-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  2. 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.

  3. Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS.

    Science.gov (United States)

    Głąb, Bartosz; Beganovic, Mirela; Anaokar, Sanket; Hao, Meng-Shu; Rasmusson, Allan G; Patton-Vogt, Jana; Banaś, Antoni; Stymne, Sten; Lager, Ida

    2016-11-25

    Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Directory of Open Access Journals (Sweden)

    John A Buglino

    2010-06-01

    Full Text Available 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.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.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.

  5. An ordered reaction mechanism for bacterial toxin acylation by the specialized acyltransferase HlyC: formation of a ternary complex with acylACP and protoxin substrates

    National Research Council Canada - National Science Library

    Stanley, Peter; Hyland, Caroline; Koronakis, Vassilis; Hughes, Colin

    1999-01-01

    .... Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis–Menten analysis...

  6. Plasma Lecithin : Cholesterol Acyltransferase Activity Is Elevated in Metabolic Syndrome and Is an Independent Marker of Increased Carotid Artery Intima Media Thickness

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; Perton, Frank; Sluiter, Wim J.; de Vries, Rindert; van Tol, Arie

    2008-01-01

    Context: Lecithin: cholesterol acyltransferase (LCAT), which esterifies free cholesterol to cholesteryl esters, is required for normal plasma lipoprotein structure and is instrumental in high density lipoprotein (HDL) remodeling, but the relationship of variation in plasma LCAT activity with

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

    OpenAIRE

    Lanfranconi, Mariana P.; Alvarez, Adri?n F; Alvarez, H?ctor M.

    2015-01-01

    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...

  8. Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed in Chinese Hamster Ovary Cells: Membrane Topology and Active Site Location

    OpenAIRE

    Lin, Song; Lu, Xiaohui; Chang, Catherine C.Y.; Chang, Ta-Yuan

    2003-01-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophi...

  9. JTP-103237, a monoacylglycerol acyltransferase inhibitor, prevents fatty liver and suppresses both triglyceride synthesis and de novo lipogenesis

    OpenAIRE

    Okuma, Chihiro; Ohta, Takeshi; Tadaki, Hironobu; Ishigure, Tatsuya; Sakata, Shohei; Taniuchi, Hideyuki; Sano, Ryuhei; Hamada, Hiromi; Kume, Shinichi; Nishiu, Jun; Kakutani, Makoto

    2015-01-01

    Aim: Monoacyglycerol acyltransferases (MGATs) are known to play important roles in intestinal TG absorption. In contrast, the role of MGATs in the liver is still unclear. We investigated the effects of JTP-103237, a novel MGAT inhibitor, on hepatic MGAT activity and hepatic lipid metabolism. Results: JTP-103237 reduced hepatic triglyceride content and hepatic MGAT activity in a high sucrose very low fat (HSVLF) diet induced fatty liver model. Interestingly, JTP-103237 suppressed not only t...

  10. Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among Populus paralogues

    Directory of Open Access Journals (Sweden)

    Johnson Virgil E

    2011-05-01

    Full Text Available Abstract Background BAHD acyltransferases are involved in the synthesis and elaboration of a wide variety of secondary metabolites. Previous research has shown that characterized proteins from this family fall broadly into five major clades and contain two conserved protein motifs. Here, we aimed to expand the understanding of BAHD acyltransferase diversity in plants through genome-wide analysis across five angiosperm taxa. We focus particularly on Populus, a woody perennial known to produce an abundance of secondary metabolites. Results Phylogenetic analysis of putative BAHD acyltransferase sequences from Arabidopsis, Medicago, Oryza, Populus, and Vitis, along with previously characterized proteins, supported a refined grouping of eight major clades for this family. Taxon-specific clustering of many BAHD family members appears pervasive in angiosperms. We identified two new multi-clade motifs and numerous clade-specific motifs, several of which have been implicated in BAHD function by previous structural and mutagenesis research. Gene duplication and expression data for Populus-dominated subclades revealed that several paralogous BAHD members in this genus might have already undergone functional divergence. Conclusions Differential, taxon-specific BAHD family expansion via gene duplication could be an evolutionary process contributing to metabolic diversity across plant taxa. Gene expression divergence among some Populus paralogues highlights possible distinctions between their biochemical and physiological functions. The newly discovered motifs, especially the clade-specific motifs, should facilitate future functional study of substrate and donor specificity among BAHD enzymes.

  11. Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among Populus paralogues

    Science.gov (United States)

    2011-01-01

    Background BAHD acyltransferases are involved in the synthesis and elaboration of a wide variety of secondary metabolites. Previous research has shown that characterized proteins from this family fall broadly into five major clades and contain two conserved protein motifs. Here, we aimed to expand the understanding of BAHD acyltransferase diversity in plants through genome-wide analysis across five angiosperm taxa. We focus particularly on Populus, a woody perennial known to produce an abundance of secondary metabolites. Results Phylogenetic analysis of putative BAHD acyltransferase sequences from Arabidopsis, Medicago, Oryza, Populus, and Vitis, along with previously characterized proteins, supported a refined grouping of eight major clades for this family. Taxon-specific clustering of many BAHD family members appears pervasive in angiosperms. We identified two new multi-clade motifs and numerous clade-specific motifs, several of which have been implicated in BAHD function by previous structural and mutagenesis research. Gene duplication and expression data for Populus-dominated subclades revealed that several paralogous BAHD members in this genus might have already undergone functional divergence. Conclusions Differential, taxon-specific BAHD family expansion via gene duplication could be an evolutionary process contributing to metabolic diversity across plant taxa. Gene expression divergence among some Populus paralogues highlights possible distinctions between their biochemical and physiological functions. The newly discovered motifs, especially the clade-specific motifs, should facilitate future functional study of substrate and donor specificity among BAHD enzymes. PMID:21569431

  12. Cloning, Characterization, and Expression Analysis of a Gene Encoding a Putative Lysophosphatidic Acid Acyltransferase from Seeds of Paeonia rockii.

    Science.gov (United States)

    Zhang, Qing-Yu; Niu, Li-Xin; Yu, Rui; Zhang, Xiao-Xiao; Bai, Zhang-Zhen; Duan, Ke; Gao, Qing-Hua; Zhang, Yan-Long

    2017-06-01

    Tree peony (Paeonia section Moutan DC.) is an excellent woody oil crop, and the cloning and functional analysis of genes related to fatty acid (FA) metabolism from this organism has not been reported. Lysophosphatidic acid acyltransferase (LPAAT), which converts lysophosphatidic acid (LPA) to phosphatidic acid (PA), catalyzes the addition of fatty acyl moieties to the sn-2 position of the LPA glycerol backbone in triacylglycerol (TAG) biosynthesis. This project reports a putative lysophosphatidic acid acyltransferase gene PrLPAAT1 isolated from Paeonia rockii. Our data indicated that PrLPAAT1 has 1047 nucleotides and encodes a putative 38.8 kDa protein with 348 amino acid residues. Bioinformatic analysis demonstrated that PrLPAAT1 contains two transmembrane domains (TMDs). Subcellular localization analysis confirmed that PrLPAAT1 is a plasma membrane protein. Phylogenetic analysis revealed that PrLPAAT1 shared 74.3 and 65.5% amino acid sequence identities with the LPAAT1 sequences from columbine and grape, respectively. PrLPAAT1 belongs to AGPAT family, and may have acyltransferase activity. PrLPAAT1 was ubiquitously expressed in diverse tissues, and PrLPAAT1 expression was higher in the flower and developing seed. PrLPAAT1 is probably an important component in the FA accumulation process, especially during the early stages of seed development. PrLPAAT1 overexpression using a seed-specific promoter increased total FA content and the main FA accumulation in Arabidopsis transgenic plants.

  13. 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. Copyright © 2015 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  14. Lecithin: cholesterol acyltransferase and na(+)-k(+)-ATPase activity in patients with breast cancer.

    Science.gov (United States)

    Ozmen, Hilal Kiziltunç; Askın, Seda

    2013-06-01

    The aim of this study was to determine whether plasma lecithin:cholesterol acyltransferase (pLCAT) and erythrocyte membrane Na(+)-K(+)-ATPase ase (emNaKATPs) activity have a correlation in breast cancer. This study compared these parameters at time points before and after treatment with radiotherapy. The levels of pLCAT and emNaKATPs were assessed in 30 patients with breast carcinoma and 20 control subjects. While emNaKATPs was measured with spectrophotometric method, pLCAT levels was measured using a specific enzyme-linked immunosorbent assay. pLCAT levels, both before and after radiotherapy, were found to be decreased in breast cancer patients than in the controls groups (p0.05). The results of the present study demonstrated that decreased pLCAT and emNaKATPs activity levels in breast cancer patients after/before RT than control group. In addition, decreased emNaKATPs activity in breast cancer patients receiving radiotherapy may be due to decreased pLCAT concentrations and RT beam. In our opinion, altered activities of pLCAT and emNaKATPs are linked to the treatment effect of radiotherapy. These data may clarify the development of cell membrane dysfunction and lipid metabolism in breast cancer patients receiving radiotherapy.

  15. 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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Binding of 8-anilino-1-naphthalenesulfonate to lecithin:cholesterol acyltransferase studied by fluorescence techniques.

    Science.gov (United States)

    Sarkar, Pabak; Bharill, Shashank; Gryczynski, Ignacy; Gryczynski, Zygmunt; Nair, Maya P; Lacko, Andras G

    2008-07-24

    The solvatochromic fluorescent probe 8-anilino-1-naphthalenesulfonate (ANS) has been used to study the hydrophobicity and conformational dynamics of lecithin:cholesterol acyltransferase (LCAT). The ANS to LCAT binding constant was estimated from titrations with ANS, keeping a constant concentration of LCAT (2 microM). Apparent binding constant was found to be dependent on the excitation. For the direct excitation of ANS at 375 nm the binding constant was 4.7 microM(-1) and for UV excitation at 295 nm was 3.2 microM(-1). In the later case, not only ANS but also tryptophan (Trp) residues of LCAT is being excited. Fluorescence spectra and intensity decays show an efficient energy transfer from tryptophan residues to ANS. The apparent distance from Trp donor to ANS acceptor, estimated from the changes in donor lifetime was about 3 nm and depends on the ANS concentration. Steady-state and time-resolved fluorescence emission and anisotropies have been characterized. The lifetime of ANS bound to LCAT was above 16 ns which is characteristic for it being in a hydrophobic environment. The ANS labeled LCAT fluorescence anisotropy decay revealed the correlation time of 42 ns with a weak residual motion of 2.8 ns. These characteristics of ANS labeled LCAT fluorescence show that ANS is an excellent probe to study conformational changes of LCAT protein and its interactions with other macromolecules.

  17. Thyrotropin and obesity: increased adipose triglyceride content through glycerol-3-phosphate acyltransferase 3.

    Science.gov (United States)

    Ma, Shizhan; Jing, Fei; Xu, Chao; Zhou, Lingyan; Song, Yongfeng; Yu, Chunxiao; Jiang, Dongqing; Gao, Ling; Li, Yujie; Guan, Qingbo; Zhao, Jiajun

    2015-01-06

    Epidemiological evidence indicates that thyrotropin (TSH) is positively correlated with the severity of obesity. However, the mechanism remains unclear. Here, we show that TSH promoted triglyceride (TG) synthesis in differentiated adipocytes in a thyroid hormone-independent manner. Mice with subclinical hypothyroidism, which is characterized by elevated serum TSH but not thyroid hormone levels, demonstrated a 35% increase in the total white adipose mass compared with their wild-type littermates. Interestingly, Tshr KO mice, which had normal thyroid hormone levels after thyroid hormone supplementation, resisted high-fat diet-induced obesity. TSH could directly induce the activity of glycerol-3-phosphate-acyltransferase 3 (GPAT3), the rate-limiting enzyme in TG synthesis, in differentiated 3T3-L1 adipocytes. However, following either the knockdown of Tshr and PPARγ or the constitutive activation of AMPK, the changes to TSH-triggered GPAT3 activity and adipogenesis disappeared. The over-expression of PPARγ or the expression of an AMPK dominant negative mutant reversed the TSH-induced changes. Thus, TSH acted as a previously unrecognized master regulator of adipogenesis, indicating that modification of the AMPK/PPARγ/GPAT3 axis via the TSH receptor might serve as a potential therapeutic target for obesity.

  18. Structural and Functional Trends in Dehydrating Bimodules from trans -Acyltransferase Polyketide Synthases

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, Drew T.; Zeng, Jia; Bailey, Constance B.; Gay, Darren C.; Yuan, Fang; Manion, Hannah R.; Keatinge-Clay, Adrian T. (Texas)

    2017-07-01

    In an effort to uncover the structural motifs and biosynthetic logic of the relatively uncharacterized trans-acyltransferase polyketide synthases, we have begun the dissection of the enigmatic dehydrating bimodules common in these enzymatic assembly lines. We report the 1.98 Å resolution structure of a ketoreductase (KR) from the first half of a type A dehydrating bimodule and the 2.22 Å resolution structure of a dehydratase (DH) from the second half of a type B dehydrating bimodule. The KR, from the third module of the bacillaene synthase, and the DH, from the tenth module of the difficidin synthase, possess features not observed in structurally characterized homologs. The DH architecture provides clues for how it catalyzes a unique double dehydration. Correlations between the chemistries proposed for dehydrating bimodules and bioinformatic analysis indicate that type A dehydrating bimodules generally produce an α/β-cis alkene moiety, while type B dehydrating bimodules generally produce an α/β-trans, γ/δ-cis diene moiety.

  19. Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2[S

    Science.gov (United States)

    Tarui, Megumi; Shindou, Hideo; Kumagai, Kazuo; Morimoto, Ryo; Harayama, Takeshi; Hashidate, Tomomi; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo; Nagase, Takahide; Shimizu, Takao

    2014-01-01

    Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo. PMID:24850807

  20. 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.

  1. Familial lecithin-cholesterol acyltransferase (LCAT) deficiency; a differential of proteinuria.

    Science.gov (United States)

    Althaf, Mohammed Mahdi; Almana, Hadeel; Abdelfadiel, Ahmed; Amer, Sadiq Mohammed; Al-Hussain, Turki Omar

    2015-01-01

    Lecithin cholesterol acyltransferase (LCAT) is an important enzyme in cholesterol metabolism that is involved in the esterification of cholesterol. A lack of this enzyme results in deranged metabolic pathways that are not completely understood, resulting in abnormal deposition of lipids in several organs. Clinically, it manifests with proteinuria, dyslipidemia and corneal opacity with progressive chronic kidney disease resulting in end-stage renal disease. We herein present a case of a 30-year-old male with proteinuria that was not responsive to empiric management with angiotensin-converting enzyme (ACE) inhibitors and oral steroids. Physical examination revealed corneal ring opacity involving both eyes. Urinalysis revealed an active sediment. The 24-h proteinuria was 3.55 grams. Family history was positive for renal disease and dyslipidemia. Viral serology for human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) were negative. Serum complements were normal and anti-nuclear antibody (ANA) was negative. We elected for a renal biopsy that revealed characteristic features of LCAT deficiency. The diagnosis of LCAT deficiency was established with a combination of clinical and pathological findings. Currently renal prognosis is poor but conservative management with ACE inhibitors and lipid lowering therapy in addition to steroids has been shown to retard progression to end-stage renal disease. However newer therapies such as gene replacement and recombinant LCAT replacement are being studied with promising preliminary results.

  2. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L..

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Chi

    Full Text Available Diacylglycerol acyltransferase (DGAT catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP. Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding.

  4. Cloning and functional analysis of three diacylglycerol acyltransferase genes from peanut (Arachis hypogaea L.).

    Science.gov (United States)

    Chi, Xiaoyuan; Hu, Ruibo; Zhang, Xiaowen; Chen, Mingna; Chen, Na; Pan, Lijuan; Wang, Tong; Wang, Mian; Yang, Zhen; Wang, Quanfu; Yu, Shanlin

    2014-01-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final and only committed acylation step in the synthesis of triacylglycerols. In this study, three novel AhDGATs genes were identified and isolated from peanut. Quantitative real-time RT-PCR analysis indicated that the AhDGAT1-2 transcript was more abundant in roots, seeds, and cotyledons, whereas the transcript abundances of AhDGAT1-1 and AhDGAT3-3 were higher in flowers than in the other tissues examined. During seed development, transcript levels of AhDGAT1-1 remained relatively low during the initial developmental stage but increased gradually during later stages, peaking at 50 days after pegging (DAP). Levels of AhDGAT1-2 transcripts were higher at 10 and 60 DAPs and much lower during other stages, whereas AhDGAT3-3 showed higher expression levels at 20 and 50 DAPs. In addition, AhDGAT transcripts were differentially expressed following exposure to abiotic stresses or abscisic acid. The activity of the three AhDGAT genes was confirmed by heterologous expression in a Saccharomyces cerevisiae TAG-deficient quadruple mutant. The recombinant yeasts restored lipid body formation and TAG biosynthesis, and preferentially incorporated unsaturated C18 fatty acids into lipids. The present study provides significant information useful in modifying the oil deposition of peanut through molecular breeding.

  5. A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins1

    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.

    2015-01-01

    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. PMID:26395841

  6. Diacylglycerol acyltransferase 1 inhibition lowers serum triglycerides in the Zucker fatty rat and the hyperlipidemic hamster.

    Science.gov (United States)

    King, Andrew J; Segreti, Jason A; Larson, Kelly J; Souers, Andrew J; Kym, Philip R; Reilly, Regina M; Zhao, Gang; Mittelstadt, Scott W; Cox, Bryan F

    2009-08-01

    Acyl CoA/diacylglycerol acyltransferase (DGAT) 1 is one of two known DGAT enzymes that catalyze the final and only committed step in triglyceride biosynthesis. The purpose of this study was to test the hypothesis that chronic inhibition of DGAT-1 with a small-molecule inhibitor will reduce serum triglyceride concentrations in both genetic and diet-induced models of hypertriglyceridemia. Zucker fatty rats and diet-induced dyslipidemic hamsters were dosed orally with A-922500 (0.03, 0.3, and 3-mg/kg), a potent and selective DGAT-1 inhibitor, for 14 days. Serum triglycerides were significantly reduced by the 3 mg/kg dose of the DGAT-1 inhibitor in both the Zucker fatty rat (39%) and hyperlipidemic hamster (53%). These serum triglyceride changes were accompanied by significant reductions in free fatty acid levels by 32% in the Zucker fatty rat and 55% in the hyperlipidemic hamster. In addition, high-density lipoprotein-cholesterol was significantly increased (25%) in the Zucker fatty rat by A-922500 administered at 3 mg/kg. This study provides the first report that inhibition of DGAT-1, the final and only committed step of triglyceride synthesis, with a selective small-molecule inhibitor, significantly reduces serum triglyceride levels in both genetic and diet-induced animal models of hypertriglyceridemia. The results of this study support further investigation of DGAT-1 inhibition as a novel therapeutic approach to the treatment of hypertriglyceridemia in humans, and they suggest that inhibition of triglyceride synthesis may have more diverse beneficial effects on serum lipid profiles beyond triglyceride lowering.

  7. Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

    Science.gov (United States)

    Kouno, Tetsuya; Akiyama, Nobuteru; Ito, Takahito; Okuda, Tomohiko; Nanchi, Isamu; Notoya, Mitsuru; Oka, Shogo; Yukioka, Hideo

    2016-02-01

    Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food. © 2016 Society for Endocrinology.

  8. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization.

    Science.gov (United States)

    Cases, S; Novak, S; Zheng, Y W; Myers, H M; Lear, S R; Sande, E; Welch, C B; Lusis, A J; Spencer, T A; Krause, B R; Erickson, S K; Farese, R V

    1998-10-09

    The synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) is an important component of cellular cholesterol homeostasis. Cholesterol ester formation also is hypothesized to be important in several physiologic processes, including intestinal cholesterol absorption, hepatic lipoprotein production, and macrophage foam cell formation in atherosclerotic lesions. Mouse tissue expression studies and the disruption of the mouse ACAT gene (Acact) have indicated that more than one ACAT exists in mammals and specifically that another enzyme is important in mouse liver and intestine. We now describe a second mammalian ACAT enzyme, designated ACAT-2, that is 44% identical to the first cloned mouse ACAT (henceforth designated ACAT-1). Infection of H5 insect cells with an ACAT-2 recombinant baculovirus resulted in expression of a approximately 46-kDa protein in cell membranes that was associated with high levels of cholesterol esterification activity. Both ACAT-1 and ACAT-2 also catalyzed the esterification of the 3beta-hydroxyl group of a variety of oxysterols. Cholesterol esterification activities for ACAT-1 and ACAT-2 exhibited different IC50 values when assayed in the presence of several ACAT-specific inhibitors, demonstrating that ACAT inhibitors can selectively target specific forms of ACAT. ACAT-2 was expressed primarily in mouse liver and small intestine, supporting the hypothesis that ACAT-2 contributes to cholesterol esterification in these tissues. The mouse ACAT-2 gene (Acact2) maps to chromosome 15 in a region containing a quantitative trait locus influencing plasma cholesterol levels. The identification and cloning of ACAT-2 will facilitate molecular approaches to understanding the role of ACAT enzymes in mammalian biology.

  9. Up-regulation of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in nephrotic syndrome.

    Science.gov (United States)

    Vaziri, Nosratola D; Liang, Kaihui

    2002-05-01

    We have previously demonstrated that hypercholesterolemia in rats with puromycin-induced nephrotic syndrome (NS) is associated with up-regulation of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and relative down-regulation of cholesterol 7alpha-hydroxylase (Ch-7alpha), which represent the rate-limiting steps in cholesterol biosynthesis and catabolism. Expression of HMG-CoA reductase is inhibited and Ch-7alpha is augmented by intracellular free cholesterol, which is avidly esterified by acyl-CoA:cholesterol acyltransferase (ACAT). Therefore, we hypothesized that NS may result in up-regulation of hepatic ACAT. Hepatic tissue ACAT mRNA (Northern blot), protein (Western blot) and enzymatic activity were determined in rats with puromycin-induced NS, placebo-treated control rats and Nagase hypoalbuminemic (NAG) rats. The NS group exhibited heavy proteinuria, hypoalbuminemia, normal creatinine clearance, severe hypercholesterolemia and hypertriglyceridemia. Despite severe hypoalbuminemia, NAG rats with inherited hypoalbuminemia exhibited only a mild elevation of plasma cholesterol and triglycerides. Severe hypercholesterolemia in the NS group was coupled with depressed liver tissue free cholesterol concentration and marked increases in hepatic ACAT mRNA, protein and enzymatic activity. In contrast, ACAT mRNA and protein contents of the liver were normal and ACAT activity was mildly elevated in the NAG group. NS results in marked up-regulation of hepatic ACAT, which is primarily due to proteinuria and not hypoalbuminemia, since the latter alone, as seen in NAG rats, does not significantly impact ACAT expression. Elevated ACAT in NS can contribute to dysregulation of cholesterol biosynthesis and catabolism by limiting the normal cholesterol signaling involved in regulation of these processes.

  10. Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation.

    Science.gov (United States)

    Toledo, Juan D; Garda, Horacio A; Cabaleiro, Laura V; Cuellar, Angela; Pellon-Maison, Magali; Gonzalez-Baro, Maria R; Gonzalez, Marina C

    2013-05-01

    Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.

  11. Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis.

    Science.gov (United States)

    Nobusawa, Takashi; Hori, Koichi; Mori, Hiroshi; Kurokawa, Ken; Ohta, Hiroyuki

    2017-05-01

    The production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large quantities of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knock-out strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-LPAT4) that catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knock-outs among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. No homologous region has been found with other lipid droplet-associated proteins, however. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by the acquisition of LPATs at the perimeter of lipid droplets. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  12. Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts.

    Science.gov (United States)

    Zienkiewicz, Krzysztof; Zienkiewicz, Agnieszka; Poliner, Eric; Du, Zhi-Yan; Vollheyde, Katharina; Herrfurth, Cornelia; Marmon, Sofia; Farré, Eva M; Feussner, Ivo; Benning, Christoph

    2017-01-01

    Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

  13. The Mitochondrial Cardiolipin Remodeling Enzyme Lysocardiolipin Acyltransferase Is a Novel Target in Pulmonary Fibrosis

    Science.gov (United States)

    Huang, Long Shuang; Mathew, Biji; Zhao, Yutong; Noth, Imre; Reddy, Sekhar P.; Harijith, Anantha; Usatyuk, Peter V.; Berdyshev, Evgeny V.; Kaminski, Naftali; Zhou, Tong; Zhang, Wei; Zhang, Yanmin; Rehman, Jalees; Kotha, Sainath R.; Gurney, Travis O.; Parinandi, Narasimham L.; Lussier, Yves A.; Garcia, Joe G. N.

    2014-01-01

    Rationale: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. Objectives: To define a role for LYCAT in human and murine models of pulmonary fibrosis. Methods: We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. Measurements and Main Results: LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. Conclusions: This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis. PMID

  14. Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus.

    Science.gov (United States)

    Murtuza, Mohammad I; Isokawa, Masako

    2018-01-01

    Ghrelin is an appetite-stimulating peptide. Serine 3 on ghrelin must be acylated by octanoate via the enzyme ghrelin-O-acyltransferase (GOAT) for the peptide to bind and activate the cognate receptor, growth hormone secretagogue receptor type 1a (GHSR1a). Interest in GHSR1a increased dramatically when GHSR1a mRNA was demonstrated to be widespread in the brain, including the cortex and hippocampus, indicating that it has multifaceted functions beyond the regulation of metabolism. However, the source of octanoylated ghrelin for GHSR1a in the brain, outside of the hypothalamus, is not well understood. Here, we report the presence of GOAT and its ability to acylate non-octanoylated ghrelin in the hippocampus. GOAT immunoreactivity is aggregated at the base of the dentate granule cell layer in the rat and wild-type mouse. This immunoreactivity was not affected by the pharmacological inhibition of GHSR1a or the metabolic state-dependent fluctuation of systemic ghrelin levels. However, it was absent in the GHSR1a knockout mouse hippocampus, pointing the possibility that the expression of GHSR1a may be a prerequisite for the production of GOAT. Application of fluorescein isothiocyanate (FITC)-conjugated non-octanoylated ghrelin in live hippocampal slice culture (but not in fixed culture or in the presence of GOAT inhibitors) mimicked the binding profile of FITC-conjugated octanoylated ghrelin, suggesting that extracellularly applied non-octanoylated ghrelin was acylated by endogenous GOAT in the live hippocampus while GOAT being mobilized out of neurons. Our results will advance the understanding for the role of endogenous GOAT in the hippocampus and facilitate the search for the source of ghrelin that is intrinsic to the brain. © 2017 International Society for Neurochemistry.

  15. Familial Lecithin:Cholesterol Acyltransferase Deficiency: First-in-Human Treatment with Enzyme Replacement

    Science.gov (United States)

    Shamburek, Robert D.; Bakker-Arkema, Rebecca; Auerbach, Bruce J.; Krause, Brian R.; Homan, Reynold; Amar, Marcelo J.; Freeman, Lita A.; Remaley, Alan T.

    2015-01-01

    BACKGROUND Humans with familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) have extremely low or undetectable HDL-C levels and by early adulthood develop many manifestations of the disorder, including corneal opacities, anemia, and renal disease. OBJECTIVE To determine if infusions of recombinant human LCAT (rhLCAT) could reverse the anemia, halt progression of renal disease and normalize HDL in FLD. METHODS rhLCAT (ACP-501) was infused i.v. over 1 hour on 3 occasions in a dose optimization phase (0.3, 3.0, and 9.0 mg/kg), then 3.0 or 9.0 mg/kg every 1–2 weeks for 7 months in a maintenance phase. Plasma lipoproteins, lipids, LCAT levels, and several measures of renal function and other clinical labs were monitored. RESULTS LCAT concentration peaked at the end of each infusion and decreased to near baseline over 7 days. Renal function generally stabilized or improved and the anemia improved. After infusion, HDL-C rapidly increased, peaking near normal in 8–12 hours; analysis of HDL particles by various methods all revealed rapid sequential disappearance of preβ-HDL and small α-4 HDL and appearance of normal α-HDL. LDL-C increased more slowly than HDL-C. Of note, triglyceride routinely decreased after meals following infusion, in contrast to the usual post-prandial increase in the absence of rhLCAT infusion. CONCLUSIONS rhLCAT infusions were well tolerated in this first-in-human study in FLD; the anemia improved, as did most parameters related to renal function in spite of advanced disease. Plasma lipids transiently normalized, and there was rapid sequential conversion of small preβ-HDL particles to mature spherical α-HDL particles. PMID:27055967

  16. Solubilization and characterization of diacylglycerol acyltransferase from microspore-derived cultures of oilseed rape.

    Science.gov (United States)

    Little, D; Weselake, R; Pomeroy, K; Furukawa-Stoffer, T; Bagu, J

    1994-12-15

    Particulate fractions prepared from microspore-derived (MD) embryos of oilseed rape (Brassica napus L. cv. Reston) and an embryogenic MD cell suspension culture of oilseed rape (B. napus L. cv. Jet Neuf) were used as a source of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) for enzyme characterization and development of a solubilization procedure. DGAT activity in the 1500-100,000 g fraction from MD embryos was stimulated 4-5-fold by 3 to 4 mg of BSA/ml of reaction mixture. DGAT activity from MD embryos was stimulated 2-3-fold by fluoride salts and 1.4-fold by NaCl, whereas iodide salts caused substantial inhibition of enzyme activity. The effect of the various 1:1 electrolytes on enzyme activity appeared to be related more to their differential effects on solution structure rather than ionic strength. DGAT was solubilized from membranes of MD embryos and the cell suspension culture by about 80 and 50% respectively, using 2 M NaCl in 1% (w/v) octanoyl-N-methyl-glucamide (MEGA-8) (pH 8.0 buffer) at a detergent to protein ratio of 2:1. The specific activity of solubilized DGAT was about 2-fold greater than that of the particulate enzyme. The mechanism of solubilization appeared to be related to the lowering of the critical micellar concentration of MEGA-8 in the presence of NaCl. DGAT, solubilized from MD embryos, eluted with an M(r) of about 2 x 10(6) during gel-filtration chromatography on a Superose 6 column equilibrated in buffer containing 0.1% (w/v) MEGA-8. The solubilized enzyme exhibited optimal activity at pH 7. At concentrations above 2 microM acyl-CoA, the specificity of solubilized DGAT for oleoyl-CoA and palmitoyl-CoA was considerably greater than for stearoyl-CoA.

  17. 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

    autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter......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...

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

    OpenAIRE

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

    2016-01-01

    ? 2016 The Authors.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 a...

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

    OpenAIRE

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

    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 thoro...

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

    OpenAIRE

    Aym?, 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 thoro...

  1. A novel homozygous mutation causing lecithin-cholesterol acyltransferase deficiency in a proband of Romanian origin with a record of extreme gestational hyperlipidemia.

    Science.gov (United States)

    Rial-Crestelo, David; Santos-Recuero, Ildefonso; Julve, Josep; Blanco-Vaca, Francisco; Torralba, Miguel

    A patient from Romania with extraordinarily high total cholesterol levels and clinical and biochemical features consistent with familial lecithin-cholesterol acyltransferase deficiency is reported. The genetic analysis performed on our proband showed a novel homozygous mutation on codon 119 of lecithin-cholesterol acyltransferase gene that causes the substitution of glycine by aspartate. The same mutation, also in homozygosis, was observed in her older sister, whereas his brother presented it in heterozygosis. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. Glycerol-3-phosphate acyltransferase 4 is essential for the normal development of reproductive organs and the embryo in Brassica napus.

    Science.gov (United States)

    Chen, Xue; Chen, Guanqun; Truksa, Martin; Snyder, Crystal L; Shah, Saleh; Weselake, Randall J

    2014-08-01

    The enzyme sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) is involved in the biosynthesis of plant lipid poly-esters. The present study further characterizes the enzymatic activities of three endoplasmic reticulum-bound GPAT4 isoforms of Brassica napus and examines their roles in the development of reproductive organs and the embryo. All three BnGPAT4 isoforms exhibited sn-2 acyltransferase and phosphatase activities with dicarboxylic acid-CoA as acyl donor. When non-substituted acyl-CoA was used as acyl donor, the rate of acylation was considerably lower and phosphatase activity was not manifested. RNA interference (RNAi)-mediated down-regulation of all GPAT4 homologues in B. napus under the control of the napin promoter caused abnormal development of several reproductive organs and reduced seed set. Microscopic examination and reciprocal crosses revealed that both pollen grains and developing embryo sacs of the B. napus gpat4 lines were affected. The gpat4 mature embryos showed decreased cutin content and altered monomer composition. The defective embryo development further affected the oil body morphology, oil content, and fatty acid composition in gpat4 seeds. These results suggest that GPAT4 has a critical role in the development of reproductive organs and the seed of B. napus. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  4. LECITHIN: CHOLESTEROL ACYLTRANSFERASE ACTIVITY IS DECREASED IN TYPE 2 DIABETES MELLITUS

    Directory of Open Access Journals (Sweden)

    A. Ghanei

    2007-09-01

    Full Text Available Lecithin cholesterol acyltransferase (LCAT plays a major role in the removal of free cholesterol from tissues via assisting HDL-C maturation, and its activity has been proposed as the main indicator of HDL-C function. The aim of the study was to measure LCAT activity in type 2 diabetic patients and to elucidate whether LCAT is associated with metabolic control, and insulin resistance. A case-control study was conducted in Imam Khomeini Hospital during 2006, recruiting 45 type 2 diabetes mellitus patients and 45 healthy subjects. Cases and controls were matched regarding gender, age and body mass index (BMI. FBS, lipid profile, LCAT activity, HbA1C, insulin were measured and insulin resistance (HOMA-IRwas calculated for both patients and controls. The studied variables were then compared between the two groups, and the association of LCAT activity with any of the variables was examined. Twenty-five subjects were female and 20 male both among patients and controls. Mean age of diabetics was 49.9 yrs (range, 40-64, and of controls 51.1 yrs (range, 39-64. FBS, HbA1C, HOMA-IR and TG in patients were significantly higher than controls, and HDL-C in controls was significantly higher than patients. LCAT activity of patients (73 9.1 µmol/L/h was significantly lower than that in controls (88 4.5 µmol/L/h (p<0.001. LCAT activity had significant inverse correlations with HbA1C and duration of diabetes. After multilinear regression analysis in patients, LCAT activity was only correlated with HbA1C level (ß= -0.9, p<0.001. LCAT activity had no significant association with HDL-C and HOMA-IR in any of the groups."nLCAT activity is significantly decreased in patients with type 2 diabetes compared with healthy controls, and has an inverse correlation with the magnitude of hyperglycemia.

  5. Variant Amino Acid Residues Alter the Enzyme Activity of Peanut Type 2 Diacylglycerol Acyltransferases

    Directory of Open Access Journals (Sweden)

    Ling Zheng

    2017-10-01

    Full Text Available Diacylglycerol acyltransferase (DGAT catalyzes the final step in triacylglycerol (TAG biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2 genes were cloned from the peanut cultivar ‘Luhua 14.’ Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h. Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA sequences. A representative full-length cDNA clone (AhDGAT2a was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon ‘sink’ that supports increased FA

  6. Characterisation of phospholipid: diacylglycerol acyltransferases (PDATs) from Camelina sativa and their roles in stress responses.

    Science.gov (United States)

    Yuan, Lixia; Mao, Xue; Zhao, Kui; Ji, Xiajie; Ji, Chunli; Xue, Jinai; Li, Runzhi

    2017-07-15

    As an important oilseed worldwide, Camelina sativa is being increasingly explored for its use in production of food, feed, biofuel and industrial chemicals. However, detailed mechanisms of camelina oil biosynthesis and accumulation, particularly in vegetative tissues, are understood to a very small extent. Here, we present genome-wide identification, cloning and functional analysis of phospholipid diacylglycerol acyltransferase (PDAT) in C. sativa, which catalyses the final acylation step in triacylglycerol (TAG) biosynthesis by transferring a fatty acyl moiety from a phospholipid to diacylglycerol (DAG). We identified five genes (namely CsPDAT1-A, B, and C and CsPDAT2-A and B) encoding PDATs from the camelina genome. CsPDAT1-A is mainly expressed in seeds, whereas CsPDAT1-C preferentially accumulates in flower and leaf tissues. High expression of CsPDAT2-A and CsPDAT2-B was detected in stem and root tissues, respectively. Cold stress induced upregulation of CsPDAT1-A and CsPDAT1-C expression by 3.5- and 2.5-fold, respectively, compared to the control. Salt stress led to an increase in CsPDAT2-B transcripts by 5.1-fold. Drought treatment resulted in an enhancement of CsPDAT2-A mRNAs by twofold and a reduction of CsPDAT2-B expression. Osmotic stress upregulated the expression of CsPDAT1-C by 3.3-fold. Furthermore, the cDNA clones of these CsPDAT genes were isolated for transient expression in tobacco leaves. All five genes showed PDAT enzymatic activity and substantially increased TAG accumulation in the leaves, with CsPDAT1-A showing a higher preference for ɑ-linolenic acid (18:3 ω-3). Overall, this study demonstrated that different members of CsPDAT family contribute to TAG synthesis in different tissues. More importantly, they are involved in different types of stress responses in camelina seedlings, providing new evidence of their roles in oil biosynthesis and regulation in camelina vegetative tissue. The identified CsPDATs may have practical applications in

  7. Characterisation of phospholipid: diacylglycerol acyltransferases (PDATs from Camelina sativa and their roles in stress responses

    Directory of Open Access Journals (Sweden)

    Lixia Yuan

    2017-07-01

    Full Text Available As an important oilseed worldwide, Camelina sativa is being increasingly explored for its use in production of food, feed, biofuel and industrial chemicals. However, detailed mechanisms of camelina oil biosynthesis and accumulation, particularly in vegetative tissues, are understood to a very small extent. Here, we present genome-wide identification, cloning and functional analysis of phospholipid diacylglycerol acyltransferase (PDAT in C. sativa, which catalyses the final acylation step in triacylglycerol (TAG biosynthesis by transferring a fatty acyl moiety from a phospholipid to diacylglycerol (DAG. We identified five genes (namely CsPDAT1-A, B, and C and CsPDAT2-A and B encoding PDATs from the camelina genome. CsPDAT1-A is mainly expressed in seeds, whereas CsPDAT1-C preferentially accumulates in flower and leaf tissues. High expression of CsPDAT2-A and CsPDAT2-B was detected in stem and root tissues, respectively. Cold stress induced upregulation of CsPDAT1-A and CsPDAT1-C expression by 3.5- and 2.5-fold, respectively, compared to the control. Salt stress led to an increase in CsPDAT2-B transcripts by 5.1-fold. Drought treatment resulted in an enhancement of CsPDAT2-A mRNAs by twofold and a reduction of CsPDAT2-B expression. Osmotic stress upregulated the expression of CsPDAT1-C by 3.3-fold. Furthermore, the cDNA clones of these CsPDAT genes were isolated for transient expression in tobacco leaves. All five genes showed PDAT enzymatic activity and substantially increased TAG accumulation in the leaves, with CsPDAT1-A showing a higher preference for ɑ-linolenic acid (18:3 ω-3. Overall, this study demonstrated that different members of CsPDAT family contribute to TAG synthesis in different tissues. More importantly, they are involved in different types of stress responses in camelina seedlings, providing new evidence of their roles in oil biosynthesis and regulation in camelina vegetative tissue. The identified CsPDATs may have practical

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

    NARCIS (Netherlands)

    Post, SM; Zoeteweij, JP; Bos, MHA; de Wit, ECM; Havinga, R; Kuipers, F; Princen, HMG

    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 (Cl- 1011), a novel ACAT inhibitor, on bile acid synthesis and cholesterol 7 alpha-hydroxylase in

  9. Human and rat bile acid-CoA : Amino acid N-acyltransferase are liver-specific peroxisomal enzymes: Implications for intracellular bile salt transport

    NARCIS (Netherlands)

    Pellicoro, Antonella; van den Heuvel, Fiona A. J.; Geuken, Mariska; Moshage, Han; Jansen, Peter L. M.; Faber, Klaas Nico

    Bile acid-coenzyme A:amino acid N-acyltransferase (BAAT) is the sole enzyme responsible for conjugation of primary and secondary bile acids to taurine and glycine. Previous studies indicate a peroxisomal location of BAAT in peroxisomes with variable amounts up to 95% detected in cytosolic fractions.

  10. Acute and chronic effects of a 24-hour intravenous triglyceride emulsion challenge on plasma lecithin : cholesterol acyltransferase, phospholipid transfer protein, and cholesteryl ester transfer protein activities

    NARCIS (Netherlands)

    Riemens, SC; Van Tol, A; Sluiter, WJ; Dullaart, RPF

    Lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and cholesteryl ester transfer protein (CETP) are key factors in remodeling of high density lipoproteins (HDL) and triglyceride-rich lipoproteins. We examined the effect of a large, 24 h intravenous fat load on plasma

  11. 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)

  12. Atherogenic Impact of Lecithin-Cholesterol Acyltransferase and Its Relation to Cholesterol Esterification Rate in HDL (FERHDL) and AIP [log(TG/HDL-C)] Biomarkers: The Butterfly Effect?

    Czech Academy of Sciences Publication Activity Database

    Dobiášová, Milada

    2017-01-01

    Roč. 66, č. 2 (2017), s. 193-203 ISSN 0862-8408 Institutional support: RVO:67985823 Keywords : lecithin-cholesterol acyltransferase (LCAT) * atherosclerosis * FERHDL (fractional esterification rate in HDL) * AIP (atherogenic index of plasma, log(TG/HDL-C) * biomarkers of cardiometabolic risk * lipoprotein particle size Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.461, year: 2016

  13. An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A : Isopenicillin N acyltransferase from Penicillium chrysogenum

    NARCIS (Netherlands)

    Yoshida, Hiromi; Hensgens, Charles M.H.; Laan, Jan Metske van der; Sutherland, John D.; Hart, Darren J.; Dijkstra, Bauke W.

    2005-01-01

    Acyl coenzyme A: isopenicillin N acyltransferase (AT) from Penicillium chrysogenum is an enzyme of interest for the biosynthesis of β-lactam antibiotics. Severe aggregation problems with wild type AT have, however, prevented significant progress in the structure–function analysis of this enzyme for

  14. 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

  15. Effect of growth hormone replacement therapy on plasma lecithin : cholesterol acyltransferase and lipid transfer protein activities in growth hormone-deficient adults

    NARCIS (Netherlands)

    Beentjes, JAM; van Tol, A; Sluiter, WJ; Dullaart, RPF

    The effects of growth hormone (GH) replacement on plasma lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and phospholipid transfer protein (PLTP), factors involved in high density lipoprotein (HDL) metabolism, We unknown. We carried out a 6 mouths study in 24

  16. 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

  17. Human acyl-CoA:cholesterol acyltransferase (ACAT) and its potential as a target for pharmaceutical intervention against atherosclerosis.

    Science.gov (United States)

    Chang, Catherine; Dong, Ruhong; Miyazaki, Akira; Sakashita, Naomi; Zhang, Yi; Liu, Jay; Guo, Michael; Li, Bo-Liang; Chang, Ta-Yuan

    2006-03-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty-acyl-coenzyme A. At the single-cell level, ACAT serves as a regulator of intracellular cholesterol homeostasis. In addition, ACAT supplies cholesteryl esters for lipoprotein assembly in the liver and small intestine. Under pathological conditions, the accumulation of cholesteryl esters produced by ACAT in macrophages contributes to foam cell formation, a hallmark of the early stage of atherosclerosis. Several reviews addressing various aspects of ACAT and ACAT inhibitors are available. This review briefly outlines the current knowledge on the biochemical properties of human ACATs, and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventions against atherosclerosis.

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

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    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.

  19. Acyl-coenzyme A:cholesterol acyltransferase-2 (ACAT-2) is responsible for elevated intestinal ACAT activity in diabetic rats.

    Science.gov (United States)

    Hori, Masaharu; Satoh, Maki; Furukawa, Kohichiro; Sakamoto, Yu-ichiro; Hakamata, Hideki; Komohara, Yoshihiro; Takeya, Motohiro; Sasaki, Yutaka; Miyazaki, Akira; Horiuchi, Seikoh

    2004-09-01

    Diabetes-induced dyslipidemia is seen in streptozotocin-induced diabetic rats. This is caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. Because two ACAT isozymes (ACAT-1 and ACAT-2) were identified, in the present study we determined which ACAT isozyme was involved in the elevated intestinal ACAT activity in diabetic rats. We cloned a full-length cDNA of rat ACAT-2. Its overexpression in ACAT-deficient AC29 cells demonstrated that the ACAT activity is derived from the cloned cDNA, and a 45-kDa protein of rat ACAT-2 cross-reacts with an anti-human ACAT-2 antibody. The tissue distribution of rat ACAT-2 mRNA revealed its restricted expression to liver and small intestine. Immunohistochemical analyses using an anti-human ACAT-2 antibody demonstrated that ACAT-2 is localized in villus-crypt axis of rat small intestine. The intestinal ACAT activity in diabetic rats was significantly immunodepleted by an anti-ACAT-2 antibody but not by an anti-ACAT-1 antibody. Finally, intestinal ACAT-2 in diabetic rats significantly increased at both protein and mRNA levels as compared with that in control rats. Our data demonstrate that ACAT-2 isozyme is responsible for the increased intestinal ACAT activity of diabetic rats, suggesting an important role of ACAT-2 for dyslipidemia in diabetic patients. Diabetic rats exhibit dyslipidemia caused, in part, by elevated intestinal acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity. We determined which ACAT isozyme (ACAT-1 or ACAT-2) was involved in the elevated intestinal ACAT activity in diabetic rats. We demonstrated an important role of ACAT-2, implicating its involvement in dyslipidemia in diabetic patients.

  20. 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.

  1. Tissue-specific expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro

    National Research Council Canada - National Science Library

    Uelmen, P J; Oka, K; Sullivan, M; Chang, C C; Chang, T Y; Chan, L

    1995-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the esterification of cholesterol with long chain fatty acids and is believed to play an important part in the development of atherosclerotic lesions...

  2. Hepatic Monoacylglycerol O-acyltransferase 1 as a Promising Therapeutic Target for Steatosis, Obesity, and Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Yasuhiro Hayashi

    2014-01-01

    Full Text Available Over the past decade, considerable advances have been made in the discovery of gene targets in metabolic diseases. However, in vivo studies based on molecular biological technologies such as the generation of knockout mice and the construction of short hairpin RNA vectors require considerable effort and time, which is a major limitation for in vivo functional analysis. Here, we introduce a liver-specific nonviral small interfering RNA (siRNA delivery system into rapid and efficient characterization of hepatic gene targets in metabolic disease mice. The comparative transcriptome analysis in liver between KKAy diabetic and normal control mice demonstrated that the expression of monoacylglycerol O-acyltransferase 1 (Mogat1, an enzyme involved in triglyceride synthesis and storage, was highly elevated during the disease progression. The upregulation of Mogat1 expression in liver was also found in other genetic (db/db and diet-induced obese mice. The silencing of hepatic Mogat1 via a liver-specific siRNA delivery system resulted in a dramatic improvement in blood glucose levels and hepatic steatosis as well as overweight with no apparent overall toxicities, indicating that hepatic Mogat1 is a promising therapeutic target for metabolic diseases. The integrated approach with transcriptomics and nonviral siRNA delivery system provides a blueprint for rapid drug discovery and development.

  3. 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.

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

    Science.gov (United States)

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

    2000-01-01

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

  5. 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.

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

    2016-01-01

    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. PMID:27077078

  7. JTP-103237, a monoacylglycerol acyltransferase inhibitor, prevents fatty liver and suppresses both triglyceride synthesis and de novo lipogenesis.

    Science.gov (United States)

    Okuma, Chihiro; Ohta, Takeshi; Tadaki, Hironobu; Ishigure, Tatsuya; Sakata, Shohei; Taniuchi, Hideyuki; Sano, Ryuhei; Hamada, Hiromi; Kume, Shinichi; Nishiu, Jun; Kakutani, Makoto

    2015-07-01

    Monoacyglycerol acyltransferases (MGATs) are known to play important roles in intestinal TG absorption. In contrast, the role of MGATs in the liver is still unclear. We investigated the effects of JTP-103237, a novel MGAT inhibitor, on hepatic MGAT activity and hepatic lipid metabolism. JTP-103237 reduced hepatic triglyceride content and hepatic MGAT activity in a high sucrose very low fat (HSVLF) diet induced fatty liver model. Interestingly, JTP-103237 suppressed not only triglyceride (TG) and diacylglycerol (DG) synthesis, but also fatty acid (FA) synthesis (de novo lipogenesis) in this model. JTP-103237 also suppressed lipogenesis-related gene expression, such as sterol regulatory element-binding protein 1-c. Moreover, JTP-103237 decreased plasma glucose levels and total cholesterol and reduced the accumulation of epididymal fats in HSVLF diet fed mice. In the present study, JTP-103237 prevented carbohydrate-induced fatty liver and suppressed both TG synthesis and de novo lipogenesis, suggesting MGAT inhibitor may prevent carbohydrate-induced metabolic disorders, including NAFLD, obesity and diabetes. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  8. A linoleic acid enriched diet increases serum cholesterol esterification by lecithin:cholesterol acyltransferase in meal-fed rats.

    Science.gov (United States)

    Romijn, D; Wiseman, S A; Scheek, L M; de Fouw, N J; van Tol, A

    1998-01-01

    Dietary fats are known to influence the fatty acid profile of plasma lipids, including phospholipids which are substrates of lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), an important enzyme in lipoprotein metabolism. We tested whether the dietary fatty acid profile has an effect on LCAT activity in an animal model. Rats were conditioned to eat two meals per day, which were enriched in either palmitic, oleic or linoleic acids, for 10 weeks. Serum was isolated from blood samples taken prior to the meal. The LCAT activity was determined in two ways: (1) by measuring serum cholesterol esterification rates, which are an estimate of LCAT action on endogenous lipoproteins, and (2) by measuring serum LCAT activity levels with excess exogenous substrates, an estimate of LCAT mass. Animals receiving the linoleic acid diet had lower serum concentrations of unesterified cholesterol and triglycerides, if compared with animals fed oleic acid or palmitic acid diets (p diet (p palmitic; p oleic and linoleic; p diet may cause increased metabolism of serum cholesterol by LCAT in rats. This effect is not due to elevated serum concentrations of LCAT or of its apolipoprotein activators, but most likely to changes in the chemical composition of endogenous lipoprotein substrates. It remains to be established whether the serum cholesterol esterification rates measured in vitro are related to in vivo rates of reverse cholesterol transport.

  9. The Unique Role of the ECERIFERUM2-LIKE Clade of the BAHD Acyltransferase Superfamily in Cuticular Wax Metabolism

    Science.gov (United States)

    Haslam, Tegan M.; Gerelle, Wesley K.; Graham, Sean W.; Kunst, Ljerka

    2017-01-01

    The elongation of very-long-chain fatty acids is a conserved process used for the production of many metabolites, including plant cuticular waxes. The elongation of precursors of the most abundant cuticular wax components of some plants, however, is unique in requiring ECERIFERUM2-LIKE (CER2-LIKE) proteins. CER2-LIKEs are a clade within the BAHD superfamily of acyltransferases. They are known to be required for cuticular wax production in both Arabidopsis and maize based on mutant studies. Heterologous expression of Arabidopsis and rice CER2-LIKEs in Saccharomyces cerevisiae has demonstrated that they modify the chain-length specificity of elongation when paired with particular condensing enzymes. Despite sequence homology, CER2-LIKEs are distinct from the BAHD superfamily in that they do not appear to use acyl transfer activity to fulfill their biological function. Here, we review the discovery and characterization of CER2-LIKEs, propose several models to explain their function, and explore the importance of CER2-LIKE proteins for the evolution of plant cuticles. PMID:28608803

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

    Directory of Open Access Journals (Sweden)

    Nawel Haïli

    Full Text Available 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.

  11. Dietary conjugated linoleic acid mixture affects the activity of intestinal acyl coenzyme A: cholesterol acyltransferase in hamsters.

    Science.gov (United States)

    Thomas Yeung, C H; Yang, L; Huang, Y; Wang, J; Chen, Z Y

    2000-12-01

    The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.

  12. Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

    Science.gov (United States)

    Soutar, A K; Garner, C W; Baker, H N; Sparrow, J T; Jackson, R L; Gotto, A M; Smith, L C

    1975-07-15

    The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

  13. Acyl-CoA N-acyltransferase influences fertility by regulating lipid metabolism and jasmonic acid biogenesis in cotton.

    Science.gov (United States)

    Fu, Wenfeng; Shen, Ying; Hao, Juan; Wu, Jianyong; Ke, Liping; Wu, Caiyun; Huang, Kai; Luo, Binglun; Xu, Mingfeng; Cheng, Xiaofei; Zhou, Xueping; Sun, Jie; Xing, Chaozhu; Sun, Yuqiang

    2015-07-02

    Cotton (Gossypium spp.) is an important economic crop and there is obvious heterosis in cotton, fertility has played an important role in this heterosis. However, the genes that exhibit critical roles in anther development and fertility are not well understood. Here, we report an acyl-CoA N-acyltransferase (EC2.3; GhACNAT) that plays a key role in anther development and fertility. Suppression of GhACNAT by virus-induced gene silencing in transgenic cotton (G. hirsutum L. cv. C312) resulted in indehiscent anthers that were full of pollen, diminished filaments and stamens, and plant sterility. We found GhACNAT was involved in lipid metabolism and jasmonic acid (JA) biosynthesis. The genes differentially expressed in GhACNAT-silenced plants and C312 were mainly involved in catalytic activity and transcription regulator activity in lipid metabolism. In GhACNAT-silenced plants, the expression levels of genes involved in lipid metabolism and jasmonic acid biosynthesis were significantly changed, the amount of JA in leaves and reproductive organs was significantly decreased compared with the amounts in C312. Treatments with exogenous methyl jasmonate rescued anther dehiscence and pollen release in GhACNAT-silenced plants and caused self-fertility. The GhACNAT gene may play an important role in controlling cotton fertility by regulating the pathways of lipid synthesis and JA biogenesis.

  14. Glycerol-3-phosphate acyltransferase 2 expression modulates cell roughness and membrane permeability: An atomic force microscopy study.

    Directory of Open Access Journals (Sweden)

    Elizabeth R Cattaneo

    Full Text Available In mammalian cells, de novo glycerolipid synthesis begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT. GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions, and overexpressed in several types of cancers and cancer-derived human cell lines where its expression contributes to the tumor phenotype. Using gene silencing and atomic force microscopy, we studied the correlation between GPAT2 expression and cell surface topography, roughness and membrane permeability in MDA-MB-231 cells. In addition, we analyzed the glycerolipid composition by gas-liquid chromatography. GPAT2 expression altered the arachidonic acid content in glycerolipids, and the lack of GPAT2 seems to be partially compensated by the overexpression of another arachidonic-acid-metabolizing enzyme, AGPAT11. GPAT2 expressing cells exhibited a rougher topography and less membrane damage than GPAT2 silenced cells. Pore-like structures were present only in GPAT2 subexpressing cells, correlating with higher membrane damage evidenced by lactate dehydrogenase release. These GPAT2-induced changes are consistent with its proposed function as a tumor-promoting gene, and might be used as a phenotypic differentiation marker. AFM provides the basis for the identification and quantification of those changes, and demonstrates the utility of this technique in the study of cancer cell biology.

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

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    Carlos Navarro-Retamal

    Full Text Available 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

  16. Palmitoyl acyltransferase, Zdhhc13, facilitates bone mass acquisition by regulating postnatal epiphyseal development and endochondral ossification: a mouse model.

    Directory of Open Access Journals (Sweden)

    I-Wen Song

    Full Text Available ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.

  17. 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. © 2016 American Society of Plant Biologists. All Rights Reserved.

  18. The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis1[OPEN

    Science.gov (United States)

    Petit, Johann; Mauxion, Jean-Philippe; Tai, Fabienne Wong Jun; Fich, Eric A.; Joubès, Jérôme; Rothan, Christophe

    2016-01-01

    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. PMID:27208295

  19. The Molecular Mechanism of Substrate Recognition and Catalysis of the Membrane Acyltransferase PatA from Mycobacteria.

    Science.gov (United States)

    Tersa, Montse; Raich, Lluís; Albesa-Jové, David; Trastoy, Beatriz; Prandi, Jacques; Gilleron, Martine; Rovira, Carme; Guerin, Marcelo E

    2017-12-11

    Glycolipids play a central role in a variety of important biological processes in all living organisms. PatA is a membrane acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements, and virulence factors of Mycobacterium tuberculosis. PatA catalyzes the transfer of a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to the 2-position of inositol in PIM1/PIM2. We report here the crystal structure of PatA in the presence of 6-O-palmitoyl-α-d-mannopyranoside, unraveling the acceptor binding mechanism. The acceptor mannose ring localizes in a cavity at the end of a surface-exposed long groove where the active site is located, whereas the palmitate moiety accommodates into a hydrophobic pocket deeply buried in the α/β core of the protein. Both fatty acyl chains of the PIM2 acceptor are essential for the reaction to take place, highlighting their critical role in the generation of a competent active site. By the use of combined structural and quantum-mechanics/molecular-mechanics (QM/MM) metadynamics, we unravel the catalytic mechanism of PatA at the atomic-electronic level. Our study provides a detailed structural rationale for a stepwise reaction, with the generation of a tetrahedral transition state for the rate-determining step. Finally, the crystal structure of PatA in the presence of β-d-mannopyranose and palmitate suggests an inhibitory mechanism for the enzyme, providing exciting possibilities for inhibitor design and the discovery of chemotherapeutic agents against this major human pathogen.

  20. AAV-mediated lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene replacement therapy rescues retinal degeneration in rd11 mice.

    Science.gov (United States)

    Dai, Xufeng; Han, Juanjuan; Qi, Yan; Zhang, Hua; Xiang, Lue; Lv, Jineng; Li, Jie; Deng, Wen-Tao; Chang, Bo; Hauswirth, William W; Pang, Ji-jing

    2014-03-20

    The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. The rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. The AAV8 (Y733F)-smCBA-Lpcat1 was delivered subretinally to postnatal day 14 (P14) rd11 mice in one eye only. At 10 weeks after injection, treated rd11 mice were examined by visually-guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT), and then killed for morphologic and biochemical examination. Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. The SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type LPCAT1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually-guided water maze behavioral performances were observed in treated rd11 mice. The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and, moreover, is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. To our knowledge, this is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.

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

    Science.gov (United States)

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

    2016-07-01

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

  2. Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1.

    Science.gov (United States)

    Xu, Yang; Chen, Guanqun; Greer, Michael S; Caldo, Kristian Mark P; Ramakrishnan, Geetha; Shah, Saleh; Wu, Limin; Lemieux, M Joanne; Ozga, Jocelyn; Weselake, Randall J

    2017-10-27

    The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Characterization of a Lipoyl Domain-Independent B-Cell Autoepitope on the Human Branched-Chain Acyltransferase in Primary Biliary Cirrhosis and Overlap Syndrome with Autoimmune Hepatitis

    Directory of Open Access Journals (Sweden)

    Antal Csepregi

    2003-01-01

    Full Text Available Background and aims: Antimitochondrial antibodies (AMA which recognize pyruvate acetyltransferase (PDC-E2 represent a highly diagnostic feature of primary biliary cirrhosis (PBC. The analysis of immunofluorescence (IF-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2 alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay.

  4. 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.

  5. 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.

  6. Structure of the human acyl-CoA:cholesterol acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia.

    Science.gov (United States)

    Katsuren, K; Tamura, T; Arashiro, R; Takata, K; Matsuura, T; Niikawa, N; Ohta, T

    2001-04-30

    Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification in mammalian cells. Two isoforms of ACAT have been reported to date (ACAT-1 and ACAT-2). ACAT-1 is ubiquitously expressed in tissues except the intestine. In contrast, ACAT-2 is expressed mainly in the intestine in humans. To investigate the relationship between ACAT-2 and dyslipidemia, we determined the structure of the human ACAT-2 gene and then studied the relationship between mutations of the ACAT-2 gene and dyslipidemia. To isolate human ACAT-2 genomic DNA, we designed primers based on the human ACAT-2 cDNA sequence: forward primer 5'-ACACCTCGATCTTGGTCCTGCCATA-3' and reverse primer 5'-GGAATGCAGACAGGGAGTCCT-3'. Using these primers, a human P1-derived artificial chromosome (PAC) library was screened by PCR-based procedures. Isolated PAC clones were completely digested with BamHI and subcloned into plasmid vector. Subclones that contained exons were screened by dot-blot hybridization using partial ACAT-2 cDNA fragments. The coding region of the ACAT-2 gene was encoded in 15 exons from 51 to 265 base pairs on a 21 kilobase span of genomic DNA. The exonic sequences coincided completely with that of ACAT-2 cDNA, and each exon-intron junction conserved splicing consensus sequences. Next, 187 (91 dyslipidemic and 96 normolipidemic) subjects were screened by PCR single-strand conformational polymorphism analysis of the ACAT-2 gene. Three mutations were identified by DNA sequencing: two missense mutations (E14G in exon 1 and T254I in exon 7) and a point mutation in intron 7 (-35G-->A). Mutations in exon 1 and intron 7 were not associated with plasma concentrations of lipids and apolipoproteins (apo). However, plasma apoC-III levels in T254I heterozygotes were significantly higher than those in subjects without mutation. Plasma triglyceride (TG) levels in T254I heterozygotes were similar to those in subjects without mutation. Although further studies are needed, our data suggest that ACAT-2

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

    Science.gov (United States)

    Letterie, G S

    2000-06-01

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

  8. 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

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

    Science.gov (United States)

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

    2001-01-01

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

  10. Accelerated triacylglycerol production and altered fatty acid composition in oleaginous microalga Neochloris oleoabundans by overexpression of diacylglycerol acyltransferase 2.

    Science.gov (United States)

    Klaitong, Paeka; Fa-Aroonsawat, Sirirat; Chungjatupornchai, Wipa

    2017-04-12

    Microalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis. The integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years. Our results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other

  11. Effect of Deletion of Ghrelin-O-Acyltransferase on the Pulsatile Release of Growth Hormone in Mice.

    Science.gov (United States)

    Xie, T Y; Ngo, S T; Veldhuis, J D; Jeffery, P L; Chopin, L K; Tschöp, M; Waters, M J; Tolle, V; Epelbaum, J; Chen, C; Steyn, F J

    2015-12-01

    Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of

  12. 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

  13. A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica.

    Science.gov (United States)

    Wei, Hehong; Shi, Ying; Ma, Xiaonian; Pan, Yufang; Hu, Hanhua; Li, Yantao; Luo, Ming; Gerken, Henri; Liu, Jin

    2017-01-01

    Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, Nannochloropsis oceanica, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of N. oceanica, but the functional role of DGAT genes, especially type-I DGAT (DGAT1), remains ambiguous. Nannochloropsis oceanica IMET1 possesses two DGAT1 genes: NoDGAT1A and NoDGAT1B. Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of N. oceanica. NoDGAT1A knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. NoDGAT1A overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. Interestingly, NoDGAT1A overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L-1 at the end of a 10-day batch culture, 47% greater than that of the control line. Taken together, our work demonstrates the functional role of No

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

    Science.gov (United States)

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

    2004-11-01

    This study tested the hypothesis that avasimibe, an inhibitor of acyl coenzyme A-cholesterol acyltransferase (ACAT), would improve treadmill exercise performance in patients with claudication secondary to peripheral arterial disease (PAD). Four hundred and forty-two patients with PAD (ankle-brachial index in the index leg of or =20% reduction post-exercise) were enrolled from 39 centers in the USA. Patients were randomized to receive oral avasimibe 50 mg, 250 mg, 750 mg or placebo for a treatment period of 12 months. Changes from baseline in peak walking time (PWT) using a graded treadmill protocol were compared among groups after 6 and 12 months of treatment. Individual group comparisons were considered statistically significant if p ACAT inhibitor avasimibe did not show clear evidence of benefit on treadmill exercise performance in patients with PAD, the results add to our knowledge of the impact of treatments directed at atherosclerosis on functional endpoints.

  15. Discovery of a potent and orally available acyl-CoA: cholesterol acyltransferase inhibitor as an anti-atherosclerotic agent: (4-phenylcoumarin)acetanilide derivatives.

    Science.gov (United States)

    Ogino, Masaki; Fukui, Seiji; Nakada, Yoshihisa; Tokunoh, Ryosuke; Itokawa, Shigekazu; Kakoi, Yuichi; Nishimura, Satoshi; Sanada, Tsukasa; Fuse, Hiromitsu; Kubo, Kazuki; Wada, Takeo; Marui, Shogo

    2011-01-01

    Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).

  16. 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.

  17. 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.

  18. Genome-Wide Identification of BAHD Acyltransferases and In vivo Characterization of HQT-like Enzymes Involved in Caffeoylquinic Acid Synthesis in Globe Artichoke

    Science.gov (United States)

    Moglia, Andrea; Acquadro, Alberto; Eljounaidi, Kaouthar; Milani, Anna M.; Cagliero, Cecilia; Rubiolo, Patrizia; Genre, Andrea; Cankar, Katarina; Beekwilder, Jules; Comino, Cinzia

    2016-01-01

    Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong to the large family of BAHD acyltransferases. Following a survey of the globe artichoke genome, we identified 69 BAHD proteins carrying the catalytic site (HXXXD). Their phylogenetic analysis together with another 43 proteins, from 21 species, representative of the BAHD family, highlighted their grouping in seven major clades. Nine globe artichoke acyltransferases clustered in a sub-group of Clade V, with 3 belonging to hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) and 2 to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) like proteins. We focused our attention on the former, HQT1, HQT2, and HQT3, as they are known to play a key role in CGA biosynthesis. The expression of genes coding for the three HQTs and correlation of expression with the CQA content is reported for different globe artichoke tissues. For the first time in the globe artichoke, we developed and applied the virus-induced gene silencing approach with the goal of assessing in vivo the effect of HQT1 silencing, which resulted in a marked reduction of both CGA and diCQAs. On the other hand, when the role of the three HQTs was assessed in leaves of Nicotiana benthamiana through their transient overexpression, significant increases in mono- and diCQAs content were observed. Using transient GFP fusion proteins expressed in N. benthamiana leaves we also established the sub-cellular localization of these three enzymes. PMID:27721818

  19. 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. © 2014

  20. FUNCTIONAL MSBB ACYLTRANSFERASE OF PHOTORHABDUS LUMINESCENS, REQUIRED FOR SECONDARY LIPID A ACYLATION IN GRAM-NEGATIVE BACTERIA, CONFERS RESISTANCE TO ANTI-MICROBIAL PEPTIDES

    Directory of Open Access Journals (Sweden)

    Z. Abi Khattar

    2016-06-01

    Full Text Available Abi Khattar Z., S. Gaudriault and A. Givaudan. 2016. A functional msbB acyltransferase of Photorhabdus luminescens, required for secondary lipid a acylation in gram-negative bacteria, confers resistance to anti-microbial peptides. Lebanese Science Journal, 17(1: 47-58. Lipid A is a potent endotoxin, and its fatty acids (lauric, myristic, and sometimes palmitic acid anchors lipopolysaccharide (LPS into the outer leaflet of the outer membrane of most Gram-negative bacteria. The highly anionic charge of the glucosamine lipid A moiety makes the LPS a powerful attractant for cationic antimicrobial peptides (AMPs. AMPs are major component of innate immunity that kill bacteria by permeabilization of lipid bilayers. Secondary lipid A acylation of Klebsiella pneumoniae, involving the acyltransferase LpxM (formally, msbB or WaaN that acylates (KDO2-(lauroyl-lipid IV-A with myristate during lipid A biosynthesis, has been associated with bacterial resistance to AMPs contributing to virulence in animal models. We investigated here the role of the msbB gene of the entomopathogenic bacterium Photorhabdus luminescens in AMP resistance, by functional complementation of the AMP susceptible K. pneumoniae lpxM mutant with the P. luminescens msbB gene. We showed that msbB (lpxM gene of P. luminescens is able to enhance polymyxin B, colistin and cecropin A resistance of K. pneumoniae lpxM mutant, compared to the non-complemented mutant. However, we could not obtain any msbB mutant of Photorhabdus by performing allelic exchange experiments based on positive selection of sucrose highly resistant mutants. We thus suggest that msbB-mediated Photorhabdus lipid A acylation is essential for outer membrane low-permeability and that modification of lipid A composition, fluidity and osmosis-resistance have an important role in the ability of Photorhabdus to grow in sucrose at high concentrations.

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

    Science.gov (United States)

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

    2008-01-01

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

  2. Identification and characterization of an efficient acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) gene from the microalga Chlorella ellipsoidea.

    Science.gov (United States)

    Guo, Xuejie; Fan, Chengming; Chen, Yuhong; Wang, Jingqiao; Yin, Weibo; Wang, Richard R C; Hu, Zanmin

    2017-02-21

    Oil in the form of triacylglycerols (TAGs) is quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase (DGAT) is considered the rate-limiting enzyme for TAG accumulation. Chlorella, a unicellular eukaryotic green alga, has attracted much attention as a potential feedstock for renewable energy production. However, the function of DGAT1 in Chlorella has not been reported. A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Chlorella ellipsoidea. The 2,142 bp open reading frame of this cDNA, designated CeDGAT1, encodes a protein of 713 amino acids showing no more than 40% identity with DGAT1s of higher plants. Transcript analysis showed that the expression level of CeDGAT1 markedly increased under nitrogen starvation, which led to significant triacylglycerol (TAG) accumulation. CeDGAT1 activity was confirmed in the yeast quadruple mutant strain H1246 by restoring its ability to produce TAG. Upon expression of CeDGAT1, the total fatty acid content in wild-type yeast (INVSc1) increased by 142%, significantly higher than that transformed with DGAT1s from higher plants, including even the oil crop soybean. The over-expression of CeDGAT1 under the NOS promoter in wild-type Arabidopsis thaliana and Brassica napus var. Westar significantly increased the oil content by 8-37% and 12-18% and the average 1,000-seed weight by 9-15% and 6-29%, respectively, but did not alter the fatty acid composition of the seed oil. The net increase in the 1,000-seed total lipid content was up to 25-50% in both transgenic Arabidopsis and B. napus. We identified a gene encoding DGAT1 in C. ellipsoidea and confirmed that it plays an important role in TAG accumulation. This is the first functional analysis of DGAT1 in Chlorella. This information is important for understanding lipid synthesis and accumulation in Chlorella and for genetic engineering to enhance oil production in microalgae

  3. 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

  4. Evidence that diacylglycerol acyltransferase 1 (DGAT1) has dual membrane topology in the endoplasmic reticulum of HepG2 cells.

    Science.gov (United States)

    Wurie, Haja R; Buckett, Linda; Zammit, Victor A

    2011-10-21

    Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1(-/-) mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.

  5. Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers.

    Science.gov (United States)

    Bussières, Sylvain; Cantin, Line; Desbat, Bernard; Salesse, Christian

    2012-02-21

    Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.

  6. 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.

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

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    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.

  8. Tomatidine, a tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting acyl-CoA:cholesterol acyl-transferase (ACAT).

    Science.gov (United States)

    Fujiwara, Yukio; Kiyota, Naoko; Tsurushima, Keiichiro; Yoshitomi, Makiko; Horlad, Hasita; Ikeda, Tsuyoshi; Nohara, Toshihiro; Takeya, Motohiro; Nagai, Ryoji

    2012-03-14

    It was previously revealed that esculeoside A, a new glycoalkaloid, and esculeogenin A, a new aglycon of esculeoside A, contained in ripe tomato ameliorate atherosclerosis in apoE-deficent mice. This study examined whether tomatidine, the aglycone of tomatine, which is a major tomato glycoalkaloid, also shows similar inhibitory effects on cholesterol ester (CE) accumulation in human monocyte-derived macrophages (HMDM) and atherogenesis in apoE-deficient mice. Tomatidine significantly inhibited the CE accumulation induced by acetylated LDL in HMDM in a dose-dependent manner. Tomatidine also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-CoA:cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that tomatidine suppresses both ACAT-1 and ACAT-2 activities. Furthermore, the oral administration of tomatidine to apoE-deficient mice significantly reduced levels of serum cholesterol, LDL-cholesterol, and areas of atherosclerotic lesions. The study provides the first evidence that tomatidine significantly suppresses the activity of ACAT and leads to reduction of atherogenesis.

  9. Enhanced root growth in phosphate-starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2).

    Science.gov (United States)

    Angkawijaya, Artik Elisa; Nguyen, Van Cam; Nakamura, Yuki

    2017-09-01

    Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non-phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid-to-galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum-localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2-overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation. © 2017 John Wiley & Sons Ltd.

  10. Characterization and Functional Analysis of a Type 2 Diacylglycerol Acyltransferase (DGAT2) Gene from Oil Palm (Elaeis guineensis Jacq.) Mesocarp in Saccharomyces cerevisiae and Transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Jin, Yuanhang; Yuan, Yijun; Gao, Lingchao; Sun, Ruhao; Chen, Lizhi; Li, Dongdong; Zheng, Yusheng

    2017-01-01

    Oil palm (Elaeis guineensis Jacq.) is the highest oil-yielding plant in the world, storing 90 and 60% (dry weight) oil in its mesocarp and kernel, respectively. To gain insights into the oil accumulation mechanism, one of the key enzymes involved in triacylglycerol (TAG) biosynthesis, a Type 2 diacylglycerol acyltransferase (DGAT2) from oil palm, was characterized for its in vivo activity. EgDGAT2 is highly expressed in mesocarp during the last two developmental stages while large amounts of oil are accumulated at the highest rate during ripening. Heterologous expression of EgDGAT2 in mutant yeast H1246 restored TAG biosynthesis with substrate preference toward unsaturated fatty acids (FAs) (16:1 and 18:1). Furthermore, seed-specific overexpression of EgDGAT2 in Arabidopsis thaliana enhanced the content of polyunsaturated FAs 18:2 and 18:3 (each by 6 mol%) in seed TAGs, when compared to that from wild-type Arabidopsis. In turn, the proportion of 18:0 and 20:0 FAs in seed TAGs from EgDGAT2 transgenic lines decreased accordingly. These results provide new insights into understanding the in vivo activity of EgDGAT2 from oil palm mesocarp, which will be of importance for metabolic enhancement of unsaturated FAs production.

  11. 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

  12. Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production.

    Science.gov (United States)

    Yuzawa, Satoshi; Deng, Kai; Wang, George; Baidoo, Edward E K; Northen, Trent R; Adams, Paul D; Katz, Leonard; Keasling, Jay D

    2017-01-20

    Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.

  13. Characterization and Functional Analysis of a Type 2 Diacylglycerol Acyltransferase (DGAT2 Gene from Oil Palm (Elaeis guineensis Jacq. Mesocarp in Saccharomyces cerevisiae and Transgenic Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yuanhang Jin

    2017-10-01

    Full Text Available Oil palm (Elaeis guineensis Jacq. is the highest oil-yielding plant in the world, storing 90 and 60% (dry weight oil in its mesocarp and kernel, respectively. To gain insights into the oil accumulation mechanism, one of the key enzymes involved in triacylglycerol (TAG biosynthesis, a Type 2 diacylglycerol acyltransferase (DGAT2 from oil palm, was characterized for its in vivo activity. EgDGAT2 is highly expressed in mesocarp during the last two developmental stages while large amounts of oil are accumulated at the highest rate during ripening. Heterologous expression of EgDGAT2 in mutant yeast H1246 restored TAG biosynthesis with substrate preference toward unsaturated fatty acids (FAs (16:1 and 18:1. Furthermore, seed-specific overexpression of EgDGAT2 in Arabidopsis thaliana enhanced the content of polyunsaturated FAs 18:2 and 18:3 (each by 6 mol% in seed TAGs, when compared to that from wild-type Arabidopsis. In turn, the proportion of 18:0 and 20:0 FAs in seed TAGs from EgDGAT2 transgenic lines decreased accordingly. These results provide new insights into understanding the in vivo activity of EgDGAT2 from oil palm mesocarp, which will be of importance for metabolic enhancement of unsaturated FAs production.

  14. 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase.

    Science.gov (United States)

    Kinnunen, P M; Klopf, F H; Bastiani, C A; Gelfman, C M; Lange, L G

    1990-02-13

    Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

  15. Association of lecithin-cholesterol acyltransferase activity measured as a serum cholesterol esterification rate and low-density lipoprotein heterogeneity with cardiovascular risk: a cross-sectional study.

    Science.gov (United States)

    Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

    2016-06-01

    The cholesterol-esterifying enzyme, lecithin-cholesterol acyltransferase (LCAT), is believed to play a key role in reverse cholesterol transport. However, recent investigations have demonstrated that higher LCAT activity levels increase the formation of triglyceride (TG)-rich lipoproteins (TRLs) and atherogenesis. We hypothesized that higher LCAT activity measured as a serum cholesterol esterification rate by the endogenous substrate method might increase the formation of TRLs and thereby alter low-density lipoprotein (LDL) heterogeneity. The estimated LDL particle size [relative LDL migration (LDL-Rm)] was measured by polyacrylamide gel electrophoresis with the LipoPhor system (Joko, Tokyo, Japan) in 538 consecutive patients with at least risk factor for atherosclerosis. Multivariate regression analysis after adjustments for traditional risk factors identified elevated TRL-related marker (TG, remnant-like particle cholesterol, apolipoprotein C-II, and apolipoprotein C-III) levels as independent predictors of smaller-sized LDL particle size, both in the overall subject population and in the subset of patients with serum LDL cholesterol levels of LDL-Rm value of ≥0.40, which suggests the presence of large amounts of small-dense LDL. The results lend support to the hypothesis that increased LCAT activity may be associated with increased formation of TRLs, leading to a reduction in LDL particle size. Therefore, to reduce the risk of atherosclerotic cardiovascular disease, it may be of importance to pay attention not only to a quantitative change in the serum LDL-C, but also to the LCAT activity which is possibly associated with LDL heterogeneity.

  16. Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

    Science.gov (United States)

    Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

    2013-10-05

    Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

  17. 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.

  18. Investigating the allosterism of acyl-CoA:cholesterol acyltransferase (ACAT) by using various sterols: in vitro and intact cell studies.

    Science.gov (United States)

    Liu, Jay; Chang, Catherine C Y; Westover, Emily J; Covey, Douglas F; Chang, Ta-Yuan

    2005-10-15

    ACAT1 (acyl-CoA:cholesterol acyltransferase 1) is thought to have two distinct sterol-binding sites: a substrate-binding site and an allosteric-activator site. In the present work, we investigated the structural features of various sterols as substrates and/or activators in vitro. The results show that without cholesterol, the plant sterol sitosterol is a poor substrate for ACAT. In the presence of cholesterol, ACAT1-mediated esterification of sitosterol is highly activated while ACAT2-mediated esterification of sitosterol is only moderately activated. For ACAT1, we show that the stereochemistry of the 3-hydroxy group at steroid ring A is a critical structural feature for a sterol to serve as a substrate, but less critical for activation. Additionally, enantiomeric cholesterol, which has the same biophysical properties as cholesterol in membranes, fails to activate ACAT1. Thus ACAT1 activation by cholesterol is the result of stereo-specific interactions between cholesterol and ACAT1, and is not related to the biophysical properties of phospholipid membranes. To demonstrate the relevance of the ACAT1 allosteric model in intact cells, we showed that sitosterol esterification in human macrophages is activated upon cholesterol loading. We further show that the activation is not due to an increase in ACAT1 protein content, but is partly due to an increase in the cholesterol content in the endoplasmic reticulum where ACAT1 resides. Together, our results support the existence of a distinct sterol-activator site in addition to the sterol-substrate site of ACAT1 and demonstrate the applicability of the ACAT1 allosteric model in intact cells.

  19. Relationships between lipophilicity and biological activities in a series of indoline-based anti-oxidative acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors.

    Science.gov (United States)

    Takahashi, Kenji; Kunishiro, Kazuyoshi; Kasai, Masayasu; Miike, Tomohiro; Kurahashi, Kazuyoshi; Shirahase, Hiroaki

    2008-01-01

    A novel series of 1-alkyl-7-amido-indoline-based anti-oxidative acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors have been reported and are expected to lower plasma cholesterol levels due to the inhibition of intestinal and hepatic ACAT, and to inhibit cholesterol accumulation in macrophages due to the inhibition of low density lipoprotein (LDL) oxidation. In the present study, relationships between lipophilicity and biological activities were examined in 13 derivatives. Lipophilicity (logP) increased and water solubility decreased with dependence on the number of carbons in the 1-alkyl chain. Inhibitory activity against both in vitro intestinal ACAT and LDL oxidation positively correlated with logP; however, the optimum logP, at which the level of activity is maximal, differed between these two effects. Inhibitory activity against in vitro plasma oxidation was weakly dependent on logP. Plasma concentrations of the derivatives after oral administration at 10 mg/kg correlated negatively with logP and positively with water solubility. Hypocholesterolemic activity in rats fed a high-cholesterol diet, and the ratio of Cmax and IC50 values for ACAT inhibition, an index of effective plasma concentration, positively and highly correlated with logP, while ex vivo inhibitory activity against plasma oxidation in rats, and the ratio of Cmax and IC50 values for the inhibition of plasma oxidation negatively correlated with logP. In conclusion, in vitro ACAT inhibitory and anti-oxidative activity were differently dependent on logP, and intestinal absorption was inversely dependent on lipophilicity in indoline-based anti-oxidative ACAT inhibitors. The hypocholesterolemic effect positively correlated and the ex vivo anti-oxidative effect negatively correlated with lipophilicity. Optimum logP as a bioavailable dual inhibitor against in vivo ACAT and lipid peroxidation was estimated to be 3.8 (1-pentyl and 1-isopentyl derivatives) in the present series of derivatives.

  20. Hypolipidemic and antioxidant activity of the novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor KY-455 in rabbits and hamsters.

    Science.gov (United States)

    Nakamura, Shohei; Kamiya, Shoji; Shirahase, Hiroaki; Kanda, Mamoru; Yoshimi, Akihisa; Tarumi, Tadatsugu; Kurahashi, Kazuyoshi

    2004-01-01

    The hypolipidemic and antioxidant effects of N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide (CAS 178469-71-1, KY-455), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, were examined in hyperlipidemic rabbits and normolipidemic hamsters. KY-455 inhibited rabbit intestinal, hepatic, macrophage and adrenal ACAT with IC50 values of 0.4, 0.9, 2.9 and 4.1 micromol/l, respectively. KY-455 also inhibited rabbit plasma and LDL-peroxidation (IC50: 0.4 and 1.7 micromol/l, respectively). In rabbits fed a high-cholesterol diet, treatment with KY-455 (30 mg/kg/day) for 8 days markedly lowered serum esterified, free, low-density lipoprotein (LDL)-cholesterol, and hepatic esterified cholesterol levels. KY-455 tended to inhibit ex vivo hepatic ACAT activity 5 h after the final administration. KY-455 also inhibited ex vivo peroxidation of plasma lipids 1 and 5 h after the final administration in rabbits. In normolipidemic hamsters fed a regular diet, treatment with KY-455 (30 mg/kg, twice a day) for 4 days significantly reduced serum esterified, free and LDL-cholesterol, and hepatic esterified and free cholesterol levels. A single administration of KY-455 (30 mg/kg) significantly inhibited ex vivo hepatic ACAT activity in hamsters. In conclusion, KY-455 showed in vitro inhibitory effects on LDL-peroxidation and macrophage ACAT activity at similar concentrations, and in vivo hypolipidemic and ex vivo antioxidative effects at the same dose. Long-term administration of KY-455 is expected to prevent the progress of atherosclerosis by lowering plasma lipid levels, inhibiting both LDL-oxidation and accumulation of cholesterol in macrophages.

  1. Ghrelin O-acyltransferase (GOAT) enzyme is overexpressed in prostate cancer, and its levels are associated with patient's metabolic status: Potential value as a non-invasive biomarker.

    Science.gov (United States)

    Hormaechea-Agulla, Daniel; Gómez-Gómez, Enrique; Ibáñez-Costa, Alejandro; Carrasco-Valiente, Julia; Rivero-Cortés, Esther; L-López, Fernando; Pedraza-Arevalo, Sergio; Valero-Rosa, José; Sánchez-Sánchez, Rafael; Ortega-Salas, Rosa; Moreno, María M; Gahete, Manuel D; López-Miranda, José; Requena, María J; Castaño, Justo P; Luque, Raúl M

    2016-12-01

    Ghrelin-O-acyltransferase (GOAT) is the key enzyme regulating ghrelin activity, and has been proposed as a potential therapeutic target for obesity/diabetes and as a biomarker in some endocrine-related cancers. However, GOAT presence and putative role in prostate-cancer (PCa) is largely unknown. Here, we demonstrate, for the first time, that GOAT is overexpressed (mRNA/protein-level) in prostatic tissues (n = 52) and plasma/urine-samples (n = 85) of PCa-patients, compared with matched controls [healthy prostate tissues (n = 12) and plasma/urine-samples from BMI-matched controls (n = 28), respectively]. Interestingly, GOAT levels in PCa-patients correlated with aggressiveness and metabolic conditions (i.e. diabetes). Actually, GOAT expression was regulated by metabolic inputs (i.e. In1-ghrelin, insulin/IGF-I) in cultured normal prostate cells and PCa-cell lines. Importantly, ROC-curve analysis unveiled a valuable diagnostic potential for GOAT to discriminate PCa at the tissue/plasma/urine-level with high sensitivity/specificity, particularly in non-diabetic individuals. Moreover, we discovered that GOAT is secreted by PCa-cells, and that its levels are higher in urine samples from a stimulated post-massage vs. pre-massage prostate-test. In conclusion, plasmatic GOAT levels exhibit high specificity/sensitivity to predict PCa-presence compared with other PCa-biomarkers, especially in non-diabetic individuals, suggesting that GOAT holds potential as a novel non-invasive PCa-biomarker. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol-3-phosphate dehydrogenase expression.

    Science.gov (United States)

    Chhikara, Sudesh; Abdullah, Hesham M; Akbari, Parisa; Schnell, Danny; Dhankher, Om Parkash

    2017-10-04

    Plant seed oil-based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum-derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by co-expressing the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol-3-phosphate dehydrogenase (GPD1) genes under the control of seed-specific promoters. Plants co-expressing DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1-co-expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild-type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1- and GPD1-co-expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Effect of sardine proteins on hyperglycaemia, hyperlipidaemia and lecithin:cholesterol acyltransferase activity, in high-fat diet-induced type 2 diabetic rats.

    Science.gov (United States)

    Benaicheta, Nora; Labbaci, Fatima Z; Bouchenak, Malika; Boukortt, Farida O

    2016-01-14

    Type 2 diabetes (T2D) is a major risk factor of CVD. The effects of purified sardine proteins (SP) were examined on glycaemia, insulin sensitivity and reverse cholesterol transport in T2D rats. Rats fed a high-fat diet (HFD) for 5 weeks, and injected with a low dose of streptozotocin, were used. The diabetic rats were divided into four groups, and they were fed casein (CAS) or SP combined with 30 or 5% lipids, for 4 weeks. HFD-induced hyperglycaemia, insulin resistance and hyperlipidaemia in rats fed HFD, regardless of the consumed protein. In contrast, these parameters lowered in rats fed SP combined with 5 or 30% lipids, and serum insulin values reduced in SP v. CAS. HFD significantly increased total cholesterol and TAG concentrations in the liver and serum, whereas these parameters decreased with SP, regardless of lipid intake. Faecal cholesterol excretion was higher with SP v. CAS, combined with 30 or 5% lipids. Lecithin:cholesterol acyltransferase (LCAT) activity and HDL3-phospholipids (PL) were higher in CAS-HF than in CAS, whereas HDL2-cholesteryl esters (CE) were lower. Otherwise, LCAT activity and HDL2-CE were higher in the SP group than in the CAS group, whereas HDL3-PL and HDL3-unesterified cholesterol were lower. Moreover, LCAT activity lowered in the SP-HF group than in the CAS-HF group, when HDL2-CE was higher. In conclusion, these results indicate the potential effects of SP to improve glycaemia, insulin sensitivity and reverse cholesterol transport, in T2D rats.

  4. 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.

  5. Integrative genomic and proteomic analyses identifies glycerol-3-phosphate acyltransferase as a target of low-dose ionizing radiation in EBV infected-B cells.

    Science.gov (United States)

    Tabe, Yoko; Hatanaka, Yasuhito; Nakashiro, Mayumi; Sekihara, Kazumasa; Yamamoto, Shinichi; Matsushita, Hiromichi; Kazuno, Saiko; Fujimura, Tsutomu; Ikegami, Takako; Nakanaga, Keita; Matsumoto, Hirotaka; Ueno, Takashi; Aoki, Junken; Yokomizo, Takehiko; Konopleva, Marina; Andreeff, Michael; Miida, Takashi; Iwabuchi, Kazuhisa; Sasai, Keisuke

    2016-01-01

    We sought to gain a better understanding of the low-dose ionizing radiation (LDIR)-induced molecular changes in transformed pre-malignant cells in their microenvironment. The cellular response to LDIR was compared and contrasted using immortalized human Epstein-Barr virus-infected B-cells (EBV-B) in mono-culture, co-culture with human bone marrow derived stromal cells (MSC), or under the LDIR-induced bystander effect. The resulting alterations in protein and gene expression (including microRNA, miRNA) were evaluated by isobaric tags for relative and absolute quantification (iTRAQ) proteomics assay, western blot, cDNA array and quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. The miRNAs let7a, miR-15b, miR-16, and miR-21, and a lipid metabolic miRNA hub miR-23b, were upregulated after LDIR exposure in the mono-cultured EBV-B cells, but were downregulated in EBV-B cells co-irradiated with MSC. A lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase, the common target of these miRNA, was downregulated at the level of protein and mRNA expression in the LDIR-exposed, mono-cultured EBV-B cells and upregulated MSC co-cultured EBV-B cells. These results suggest a putative miRNA regulatory mechanism controlling the LDIR-induced stress response, and illustrate that LDIR exposure, and the cell's microenvironment, can affect specific gene expression, both directly and indirectly, resulting in altered protein expression.

  6. Structure of the Bifunctional Acyltransferase/Decarboxylase LnmK from the Leinamycin Biosynthetic Pathway Revealing Novel Activity for a Double-Hot-Dog Fold

    Energy Technology Data Exchange (ETDEWEB)

    Lohman, Jeremy R. [Scripps Research Inst., Jupiter, FL (United States); Bingman, Craig A. [Univ. of Wisconsin, Madison, WI (United States); Phillips Jr., George N. [Univ. of Wisconsin, Madison, WI (United States); Rice Univ., Houston, TX (United States); Shen, Ben [Scripps Research Inst., Jupiter, FL (United States)

    2013-01-15

    The β-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900–6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of the monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. But, to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. Moreover, LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.

  7. 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. © 2016 American Society of Plant Biologists. All Rights Reserved.

  8. 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.

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

    Science.gov (United States)

    Shen, Bo; Damude, Howard G.; Everard, John D.; Booth, John R.

    2016-01-01

    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. PMID:27208257

  10. Novel missense variants in LCAT and APOB genes in an Italian kindred with familial lecithin:cholesterol acyltransferase deficiency and hypobetalipoproteinemia

    Science.gov (United States)

    Conca, Paola; Pileggi, Silvana; Simonelli, Sara; Boer, Emanuela; Boscutti, Giuliano; Magnolo, Lucia; Tarugi, Patrizia; Penco, Silvana; Franceschini, Guido; Calabresi, Laura; Gomaraschi, Monica

    2012-01-01

    Background Lecithin:cholesterol acyltransferase (LCAT) is responsible for cholesterol esterification in plasma. Mutations of LCAT gene cause familial LCAT deficiency, a metabolic disorder characterized by hypoalphalipoproteinemia. Apolipoprotein B (apoB) is the main protein component of very-low-density lipoproteins and low-density lipoprotein (LDL). Mutations of APOB gene cause familial hypobetalipoproteinemia, a codominant disorder characterized by low plasma levels of LDL cholesterol and apoB. Objective This was a genetic and biochemical analysis of an Italian kindred with hypobetalipoproteinemia whose proband presented with hypoalphalipoproteinemia and severe chronic kidney disease. Methods Plasma lipids and apolipoproteins, cholesterol esterification, and high-density lipoprotein (HDL) subclass distribution were analyzed. LCAT and APOB genes were sequenced. Results The proband had severe impairment of plasma cholesterol esterification and high preβ-HDL content. He was heterozygote for the novel LCAT P406L variant, as were two other family members. The proband’s wife and children presented with familial hypobetalipoproteinemia and were heterozygotes for the novel apoB H1401R variant. Cholesterol esterification rate of apoB H1401R carriers was reduced, likely attributable to the low amount of circulating LDL. After renal transplantation, proband’s lipid profile, HDL subclass distribution, and plasma cholesterol esterification were almost at normal levels, suggesting a mild contribution of the LCAT P406L variant to his pretransplantation severe hypoalphalipoproteinemia and impairment of plasma cholesterol esterification. Conclusion LCAT P406L variant had a mild effect on lipid profile, HDL subclass distribution, and plasma cholesterol esterification. ApoB H1401R variant was identified as possible cause of familial hypobetalipoproteinemia and resulted in a reduction of cholesterol esterification rate. PMID:22658148

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

    Science.gov (United States)

    Gahete, Manuel D; Córdoba-Chacón, José; Hergueta-Redondo, Marta; Martínez-Fuentes, Antonio J; Kineman, Rhonda D; Moreno-Bueno, Gema; Luque, Raúl M; Castaño, Justo P

    2011-01-01

    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.

  12. 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.

  13. Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

    Science.gov (United States)

    2013-01-01

    Background Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. Methods We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. Results We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. Conclusions This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific

  14. A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

    Science.gov (United States)

    Gahete, Manuel D.; Córdoba-Chacón, José; Hergueta-Redondo, Marta; Martínez-Fuentes, Antonio J.; Kineman, Rhonda D.; Moreno-Bueno, Gema

    2011-01-01

    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. PMID:21829727

  15. Mechanism for adaptive modification during cold acclimation of phospholipid acyl chain composition in Tetrahymena. II. Activities of 2-acyl-sn-glycerol-3-phosphorylcholine and 2-acyl-sn-glycerol-3- phosphorylethanolamine acyltransferases involving the reacylation.

    Science.gov (United States)

    Yoshioka, S; Kameyama, Y; Nozawa, Y

    1984-03-27

    The deacylation-reacylation process is very important for the alteration of phospholipid fatty acyl composition on lowering of growth temperature in Tetrahymena pyriformis (Kameyama, Y., Yoshioka, S. and Nozawa, Y., (1984) Biochim. Biophys. Acta 793, 28-33). Microsomes isolated from Tetrahymena cells have reacylation activities not only for 1-acyl-sn-glycerol-3-phosphorylcholine (1-acyl-GPC) and 1-acyl-sn-glycerol-3-phosphorylethanolamine (1-acyl-GPE) but also for 2-acyl-GPC and 2-acyl-GPE. Unsaturated fatty acyl-CoAs were in general much better substrates than saturated fatty acyl-CoAs for acylations of 1-acyl-GPC and 1-acyl-GPE. The acylation rates for 1-acyl-GPE were almost the same in palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA and gamma-linoleoyl-CoA. However, the acylation activity for 1-acyl-GPC was more than 2-fold higher with palmitoleoyl-CoA than with any other unsaturated fatty acyl-CoAs. In contrast, both 2-acyl-GPC and 2-acyl-GPE acyltransferases did not show a distinct preference for various acyl-CoAs, although palmitoyl-CoA was incorporated into both 2-acylphospholipids at higher rates than into 1-acylphospholipids. These specificities for various acyl-CoAs of 1-acyl- and 2-acyl-GPC and 1-acyl- and 2-acyl-GPE acyltransferases were not changed in the microsomes isolated from cells grown isothermally at 39 degrees C and 15 degrees C and cells shifted from 39 degrees C to 15 degrees C. However, the acylating ratio of linoleoyl-CoA to palmitoyl-CoA, which were chosen as typical unsaturated and saturated fatty acyl-CoAs, in the microsomes from cells grown at 15 degrees C was 1.5-3.0-times higher than in the microsomes from 39 degrees C-grown cells in four acyltransferase activities. These results suggest that the changes of acyl-CoA specificities in reacylation enzyme activities during temperature down-shift would make little contribution to the increase in unsaturated fatty acids in phospholipids, although reacylating enzymes from isothermally grown

  16. 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

    is catalyzed by acyl-CoA: ethanol O-acyltransferases Eht1 or Eeb1 in Saccharomyces cerevisiae. In this study, these two yeast enzymes were selected to explore their preparations as the form of whole cell biocatalysts for the production of volatile flavour esters. Here, the novel whole cell biocatalysts Pichia...... esters from wort medium. Interestingly, there is no significant difference between P. pastoris-EHT1 and P. pastoris-EEB1 in substrate preference during flavour biosynthesis, indicating a similar role of Eht1 and Eeb1 in P. pastoris cells, in contradiction with previous findings in S. cerevisiae to some......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...

  17. Sterol O-acyltransferase 1 (SOAT1, ACAT) is a novel target of steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) in the human adrenal.

    Science.gov (United States)

    Ferraz-de-Souza, Bruno; Hudson-Davies, Rebecca E; Lin, Lin; Parnaik, Rahul; Hubank, Mike; Dattani, Mehul T; Achermann, John C

    2011-04-01

    Steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) is a master regulator of adrenal development and steroidogenesis. Defects in several known targets of SF-1 can cause adrenal disorders in humans. We aimed to identify novel targets of SF-1 in the human adrenal. These factors could be important regulators of adrenal development and steroidogenesis and potential candidates for adrenal dysfunction. A gene discovery strategy was developed based on bidirectional manipulation of SF-1. Overexpression or knockdown of SF-1 in NCI-H295R human adrenocortical cells was used to identify a subset of positively-regulated SF-1 targets. This approach identified well-established SF-1 target genes (STAR, CYP11A) and several novel genes (VSNL1, ZIM2, PEG3, SOAT1, and MTSS1). Given its role in cholesterol metabolism, sterol O-acyltransferase 1 (SOAT1, previously referred to as acyl-Coenzyme A:cholesterol acyltransferase 1, ACAT) was studied further and found to be expressed in the developing human fetal adrenal cortex. We hypothesized that impaired SOAT1 activity could result in adrenal insufficiency through reduced cholesteryl ester reserves or through toxic destruction of the adrenal cells during development. Therefore, mutational analysis of SOAT1 in a cohort of 43 patients with unexplained adrenal insufficiency was performed but failed to reveal significant coding sequence changes. Our reverse discovery approach led to the identification of novel SF-1 targets and defined SOAT1 as an important factor in human adrenal steroidogenesis. SF-1-dependent up-regulation of SOAT1 may be important for maintaining readily-releasable cholesterol reserves needed for active steroidogenesis and during episodes of recurrent stress.

  18. Genetic and structural identification of an O-acyltransferase gene (oacC) responsible for the 3/4-O-acetylation on rhamnose III in Shigella flexneri serotype 6.

    Science.gov (United States)

    Knirel, Yuriy A; Wang, Jianping; Luo, Xia; Senchenkova, Sofya N; Lan, Ruiting; Shpirt, Anna M; Du, Pengcheng; Shashkov, Alexander S; Zhang, Nan; Xu, Jianguo; Sun, Qiangzheng

    2014-10-21

    O-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1-5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [→2)-α-l-Rhap III-(1 → 2)-α-l-Rhap II]. In serotype 6 and some non-6 serotypes, RhaIII is O-acetylated at position either 3 or 4 (3/4-O-acetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6. Using genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on RhaIII in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-O-acetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested. Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on RhaIII specific to serotype 6.

  19. 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.

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

    Science.gov (United States)

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

    1998-08-01

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

  1. Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux.

    Science.gov (United States)

    Anthanont, Pimjai; Asztalos, Bela F; Polisecki, Eliana; Zachariah, Benoy; Schaefer, Ernst J

    2015-01-01

    We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C>A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large α-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P cholesterol in the esterified form was also significantly (P values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I-mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P cholesterol and very large α-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity. Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  2. Discovery of a novel acyl-CoA: cholesterol acyltransferase inhibitor: the synthesis, biological evaluation, and reduced adrenal toxicity of (4-phenylcoumarin)acetanilide derivatives with a carboxylic acid moiety.

    Science.gov (United States)

    Ogino, Masaki; Nakada, Yoshihisa; Negoro, Nobuyuki; Itokawa, Shigekazu; Nishimura, Satoshi; Sanada, Tsukasa; Satomi, Tomoko; Kita, Shunbun; Kubo, Kazuki; Marui, Shogo

    2011-01-01

    As a part of our research for novel potent and orally available acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors that can be used as anti-atherosclerotic agents, we recently reported the discovery of the (4-phenylcoumarine)acetanilide derivative 1. However, compound 1 showed adrenal toxicity in animal models. In order to search for safer ACAT inhibitors that do not have adrenal toxicity, we examined the inhibitory activity of ACAT in human macrophage and adrenal cells. The introduction of a carboxylic acid moiety on the pendant phenyl ring and the adjustment of the lipophilicity led to the discovery of (2E)-3-[7-chloro-3-[2-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-2-oxoethyl]-6-methyl-2-oxo-2H-chromen-4-yl]phenyl]acrylic acid (21e), which showed potent ACAT inhibitory activity in macrophages and a selectivity of around 30-fold over adrenal cells. In addition, compound 21e showed high adrenal safety in guinea pigs.

  3. Dominance and parent-of-origin effects of coding and non-coding alleles at the acylCoA-diacylglycerol-acyltransferase (DGAT1 gene on milk production traits in German Holstein cows

    Directory of Open Access Journals (Sweden)

    Weikard Rosemarie

    2007-09-01

    Full Text Available Abstract Background Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1 gene by using data sets including sires with accumulated phenotypic observations of daughters (breeding values, daughter yield deviations. However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits. Results Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits. Conclusion Non-additive effects like those identified at the bovine DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits.

  4. 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.

    Science.gov (United States)

    Zhuang, Shiwen; Fu, Junshu; Powell, Chris; Huang, Jinhai; Xia, Yihe; Yan, Ruixiang

    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 is catalyzed by acyl-CoA:ethanol O-acyltransferases Eht1 or Eeb1 in Saccharomyces cerevisiae. In this study, these two yeast enzymes were selected to explore their preparations as the form of whole cell biocatalysts for the production of volatile flavour esters. Here, the novel whole cell biocatalysts Pichia pastoris yeasts with functional extracellular expression of Eht1 or Eeb1 were constructed. Flavour production was established through an integrated process with coupled enzyme formation and ester biosynthesis in the recombinant yeasts in one pot, leading to the formation of volatile C6-C14 methyl and ethyl esters from wort medium. Interestingly, there is no significant difference between P. pastoris-EHT1 and P. pastoris-EEB1 in substrate preference during flavour biosynthesis, indicating a similar role of Eht1 and Eeb1 in P. pastoris cells, in contradiction with previous findings in S. cerevisiae to some extent. Consequently the study not only provides a greater understanding of these two enzymes in a heterogeneous host, but also demonstrated the positive effect of the recombinant Eht1 and Eeb1 in ester formation by P. pastoris live cells, potentially paving the way towards achieving efficient production of volatile flavour by an integrated biocatalytic system composed of recombinant enzyme production and flavour biosynthesis.

  5. Genome-wide analysis of PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) genes in plants reveals the eudicot-wide PDAT gene expansion and altered selective pressures acting on the core eudicot PDAT paralogs.

    Science.gov (United States)

    Pan, Xue; Peng, Fred Y; Weselake, Randall J

    2015-03-01

    PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) is an enzyme that catalyzes the transfer of a fatty acyl moiety from the sn-2 position of a phospholipid to the sn-3-position of sn-1,2-diacylglyerol, thus forming triacylglycerol and a lysophospholipid. Although the importance of PDAT in triacylglycerol biosynthesis has been illustrated in some previous studies, the evolutionary relationship of plant PDATs has not been studied in detail. In this study, we investigated the evolutionary relationship of the PDAT gene family across the green plants using a comparative phylogenetic framework. We found that the PDAT candidate genes are present in all examined green plants, including algae, lowland plants (a moss and a lycophyte), monocots, and eudicots. Phylogenetic analysis revealed the evolutionary division of the PDAT gene family into seven major clades. The separation is supported by the conservation and variation in the gene structure, protein properties, motif patterns, and/or selection constraints. We further demonstrated that there is a eudicot-wide PDAT gene expansion, which appears to have been mainly caused by the eudicot-shared ancient gene duplication and subsequent species-specific segmental duplications. In addition, selection pressure analyses showed that different selection constraints have acted on three core eudicot clades, which might enable paleoduplicated PDAT paralogs to either become nonfunctionalized or develop divergent expression patterns during evolution. Overall, our study provides important insights into the evolution of the plant PDAT gene family and explores the evolutionary mechanism underlying the functional diversification among the core eudicot PDAT paralogs. © 2015 American Society of Plant Biologists. All Rights Reserved.

  6. 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.

  7. Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

    Science.gov (United States)

    Teichert, T; Wodtke, E

    1992-12-02

    Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm

  8. Effet des grignons d'olive sur l'activité de la lécithine : cholestérol acyltransférase, chez le rat soumis à un régime enrichi en cholestérol [Effect of olive cake on lecithin: cholesterol acyltransferase activity in rats fed a cholesterol-enriched diet

    Directory of Open Access Journals (Sweden)

    Sherazede BOUDERBALA

    2015-12-01

    Full Text Available Introduction. In Mediterranean areas, the olive oil industry produces substantial amounts of by-products. Olive Cake is the solid residue obtained after olive oil extraction. Objective. The effect of olive cake was studied on the amounts and composition of lipoproteins and activity of lecithin: cholesterol acyltransferase (LCAT, in rats fed a cholesterol-enriched diet. Material and methods. Male Wistar rats (n = 24 weighing 80 ± 5 g were fed a diet containing 20% casein and enriched with 1% cholesterol (HC supplemented or not with OC at 2.5%, 5% and 7.5% (HC-OC , HC-OC and HC-OC , respectively for 28 days. Results. Hypercholesterolemic rats fed diet supplemented with OC was compared to HC group. Serum total cholesterol (TC content was 1.5- 1.7- 2.1-fold lower in HC-OC , HC-OC - and HC-OC groups, whereas high density lipoproteins-cholesterol (HDL -C and HDL -C contents were significantly increased in all HC-OC groups (P<0.05. HDL amounts were significantly increased in HC-OC , HC-OC and HC-OC groups (P<0.05. HDL -TG values were 1.5-fold higher in HC-OC group. HDL amounts and phospholipids (PL contents were 1.4- and 1.8-, 1.6- and 2.6- and 1.8- and 2.8-fold increased in HC-OC , HC-OC and HC-OC groups respectively, compared to HC group. HDL -TG and CE values were significantly increased in HC-OC (P<0.05. LCAT activity was 1.5 and 2-fold higher in HC-OC and HC-OC groups, and 2.3-fold lower in HC-OC. Conclusion. It seems that olive cake supplementation at 2.5 and 5% in hypercholesterolemic rats diets is in favor of an efficient reverse cholesterol transport from peripheral tissues to liver.

  9. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    Science.gov (United States)

    2015-10-01

    23594−23600. (13) Lobo, S., Greentree, W. K., Linder, M. E., and Deschenes, R. J. (2002) Identification of a Ras palmitoyltransferase in Saccharomyces ... 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...supernatant in the presence of 10 g/ml polybrene (Millipore). Cells were incubated for 24–48 h before splitting into selection medium . Site-directed

  10. 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.

  11. 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.

  12. Carnitine-acyltransferase system inhibition, cancer cell death, and prevention of myc-induced lymphomagenesis.

    Science.gov (United States)

    Pacilli, Annalisa; Calienni, Maria; Margarucci, Sabrina; D'Apolito, Maria; Petillo, Orsolina; Rocchi, Laura; Pasquinelli, Gianandrea; Nicolai, Raffaella; Koverech, Aleardo; Calvani, Menotti; Peluso, Gianfranco; Montanaro, Lorenzo

    2013-04-03

    The metabolic alterations of cancer cells represent an opportunity for developing selective antineoplastic treatments. We investigated the therapeutic potential of ST1326, an inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme for fatty acid (FA) import into mitochondria. ST1326 was tested on in vitro and in vivo models of Burkitt's lymphoma, in which c-myc, which drives cellular demand for FA metabolism, is highly overexpressed. We performed assays to evaluate the effect of ST1326 on proliferation, FA oxidation, and FA mitochondrial channeling in Raji cells. The therapeutic efficacy of ST1326 was tested by treating Eµ-myc mice (control: n = 29; treatment: n = 24 per group), an established model of c-myc-mediated lymphomagenesis. Experiments were performed on spleen-derived c-myc-overexpressing B cells to clarify the role of c-myc in conferring sensitivity to ST1326. Survival was evaluated with Kaplan-Meier analyses. All statistical tests were two-sided. ST1326 blocked both long- and short-chain FA oxidation and showed a strong cytotoxic effect on Burkitt's lymphoma cells (on Raji cells at 72 hours: half maximal inhibitory concentration = 8.6 μM). ST1326 treatment induced massive cytoplasmic lipid accumulation, impairment of proper mitochondrial FA channeling, and reduced availability of cytosolic acetyl coenzyme A, a fundamental substrate for de novo lipogenesis. Moreover, treatment with ST1326 in Eµ-myc transgenic mice prevented tumor formation (P = .01), by selectively impairing the growth of spleen-derived primary B cells overexpressing c-myc (wild-type cells + ST1326 vs. Eµ-myc cells + ST1326: 99.75% vs. 57.5%, difference = 42.25, 95% confidence interval of difference = 14% to 70%; P = .01). Our data indicate that it is possible to tackle c-myc-driven tumorigenesis by altering lipid metabolism and exploiting the neoplastic cell addiction to FA oxidation.

  13. 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.

  14. Rat liver dihydroxyacetone-phosphate acyltransferase : Enzyme characteristics and localization studies

    NARCIS (Netherlands)

    Hardeman, D.; Bosch, H. van den

    1988-01-01

    Peroxisomes were isolated from rat liver by pelleting a light mitochondrial (L) fraction over a 30% (w/v) Metrizamide layer. Peroxisomes were recovered as a loose pellet from the bottom of the tube and the purity of the peroxisomal fraction was calculated to be about 90%. The characteristics of

  15. 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

  16. 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...

  17. 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

    -ATs with unusual extender unit specificities. Currently, the best-studied trans-AT with nonmalonyl specificity is KirCII from kirromycin biosynthesis. Here, we eveloped an assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues...

  18. 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

    to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates...

  19. Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Beatriz Lázaro

    Full Text Available Triglycerides (TAGs, the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT. With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

  20. The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases.

    Science.gov (United States)

    Lo Leggio, Leila; Dal Degan, Florence; Poulsen, Peter; Andersen, Søren Møller; Larsen, Sine

    2003-05-13

    The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.

  1. Synthesis of a novel series of 2-alkylthio substituted naphthoquinones as potent acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors.

    Science.gov (United States)

    Lee, Kyeong; Cho, Soo Hyun; Lee, Jee Hyun; Goo, Jail; Lee, Sung Yoon; Boovanahalli, Shanthaveerappa K; Yeo, Siok Koon; Lee, Sung-Joon; Kim, Young Kook; Kim, Dong Hee; Choi, Yongseok; Song, Gyu-Yong

    2013-04-01

    We report a new series of naphthoquinone derivatives as potent ACAT inhibitors, which were obtained through structural variations of previously disclosed lead 1. Several analogs represented by 3i-l, 4k-m, 6a-n, 7a, and 7i demonstrated potent human macrophage ACAT inhibitory activity by a cell-based reporter assay with human HepG2 cell lines. In particular, compounds 4l and 6j emerged as highly potent inhibitors, exhibiting significantly high inhibitory potencies with IC50 values of 0.44 μM and 0.6 μM, respectively. Moreover, compound 4l significantly reduced the accumulation of cellular cholesterol in HepG2 cell lines. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. The effect of malonyl-CoA on overt and latent carnitine acyltransferase activities in rat liver and adipocyte mitochondria.

    OpenAIRE

    Saggerson, E D; Carpenter, C A

    1983-01-01

    1. Carnitine palmitoyltransferase and carnitine octanoyltransferase activities were measured in mitochondria at various acyl-CoA concentrations before and after sonication, thus permitting assessment of both overt and latent activities. 2. Overt carnitine palmitoyltransferase in liver and adipocyte mitochondria and overt carnitine octanoyltransferase in liver mitochondria were inhibited by malonyl-CoA. None of the latent activities were affected by this metabolite. 3. 5,5'-Dithiobis-(2-nitrob...

  3. JTP-103237, a monoacylglycerol acyltransferase inhibitor, prevents fatty liver and suppresses both triglyceride synthesis and de novo lipogenesis

    Directory of Open Access Journals (Sweden)

    Chihiro Okuma

    2015-07-01

    Conclusion: In the present study, JTP-103237 prevented carbohydrate-induced fatty liver and suppressed both TG synthesis and de novo lipogenesis, suggesting MGAT inhibitor may prevent carbohydrate-induced metabolic disorders, including NAFLD, obesity and diabetes.

  4. Identification and Characterization of Two Klebsiella pneumoniae lpxL Lipid A Late Acyltransferases and Their Role in Virulence.

    Science.gov (United States)

    Mills, Grant; Dumigan, Amy; Kidd, Timothy; Hobley, Laura; Bengoechea, José A

    2017-09-01

    Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniaelpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs. Copyright © 2017 American Society for Microbiology.

  5. Mice with alopecia, osteoporosis, and systemic amyloidosis due to mutation in Zdhhc13, a gene coding for palmitoyl acyltransferase.

    Directory of Open Access Journals (Sweden)

    Amir N Saleem

    2010-06-01

    Full Text Available Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein-protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea-mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions. Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13, a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X, and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  7. Protein (Cyanobacteria): 176658 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available xymyristoyl) glucosamine N-acyltransferase Cyanobium gracile PCC 6307 MRFSSLLTRIGAVQDGSPRHLAGDPEITGAEALDRAGP...GVVLYEDVQLGEGCEIHANAVLHPGSRLGAGCVIQSQAVIGAEGFGFVPTATGWVKMPQTGRVVLEDGVEVGCGSTIDRPSVGETRIGA

  8. 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....

  9. Purification, crystallization and preliminary X-ray diffraction of Cys103Ala acyl coenzyme A : isopenicillin N acyltransferase from Penicillium chrysogenum

    NARCIS (Netherlands)

    Hensgens, Charles M.H.; Kroezinga, Els A.; Montfort, Bart A. van; Laan, Jan-Metske van der; Sutherland, John D.; Dijkstra, Bauke W.

    2002-01-01

    Penicillins and cephalosporins are an efficacious group of antibiotics produced by fungi such as Penicillium chrysogenum and Acremonium chrysogenum. The last step in their biosynthesis is catalyzed by acyl coenzyme A:isopenicillin N transferase (AT). This enzyme is produced as a single-chain

  10. Synthesis and structure-activity relationship studies on a novel series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT).

    Science.gov (United States)

    Ohnuma, Satoshi; Muraoka, Masami; Ioriya, Katsuhisa; Ohashi, Naohito

    2004-03-08

    The synthesis and structure-activity relationships of N-phenyl-N'-[3-(4-phenylnaphthylidinoyl)]urea derivatives 3 as a novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring, together with a 1-N-(n)butyl substitution, SM-32504 (3m), gave a potent ACAT inhibitor, in vitro, respectively. The most potent compound, SM-32504 (3m), decreased the serum cholesterol level significantly in a high fat and high cholesterol-fed mouse model.

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

    Science.gov (United States)

    Li, L; Pownall, H J

    2000-12-01

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

  12. Acyl-CoA:cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowering effect in ApoE*3-Leiden mice

    NARCIS (Netherlands)

    Delsing, D. J.; Offerman, E. H.; van Duyvenvoorde, W.; van der Boom, H.; de Wit, E. C.; Gijbels, M. J.; van der Laarse, A.; Jukema, J. W.; Havekes, L. M.; Princen, H. M.

    2001-01-01

    The present study investigated whether the ACAT inhibitor avasimibe can reduce atherogenesis independently of its cholesterol-lowering effect in ApoE*3-Leiden mice. Two groups of 15 female ApoE*3-Leiden mice were put on a high-cholesterol (HC) diet; 1 group received 0.01% (wt/wt) avasimibe mixed

  13. A Novel Human Ghrelin Variant (In1-Ghrelin) and Ghrelin-O-Acyltransferase Are Overexpressed in Breast Cancer: Potential Pathophysiological Relevance

    OpenAIRE

    Gahete, Manuel D.; José Córdoba-Chacón; Marta Hergueta-Redondo; Martínez-Fuentes, Antonio J.; Kineman, Rhonda D.; Gema Moreno-Bueno; Luque, Raúl M.; Castaño, Justo P.

    2011-01-01

    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 var...

  14. Genome-wide identification of bahd acyltransferases and in vivo characterization of HQT-like enzymes involved in caffeoylquinic acid synthesis in globe artichoke

    NARCIS (Netherlands)

    Moglia, Andrea; Acquadro, Alberto; Eljounaidi, Kaouthar; Milani, Anna M.; Cagliero, Cecilia; Rubiolo, Patrizia; Genre, Andrea; Cankar, Katarina; Beekwilder, Jules; Comino, Cinzia

    2016-01-01

    Globe artichoke (Cynara cardunculus L. var. scolymus) is a rich source of compounds promoting human health (phytonutrients), among them caffeoylquinic acids (CQAs), mainly represented by chlorogenic acid (CGA), and dicaffeoylquinic acids (diCQAs). The enzymes involved in their biosynthesis belong

  15. A high-throughput-compatible fluorescence anisotropy-based assay for competitive inhibitors of Escherichia coli UDP-N-acetylglucosamine acyltransferase (LpxA).

    Science.gov (United States)

    Shapiro, Adam B; Ross, Philip L; Gao, Ning; Livchak, Stephania; Kern, Gunther; Yang, Wei; Andrews, Beth; Thresher, Jason

    2013-03-01

    LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.

  16. 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

    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...

  17. Des-Acyl Ghrelin and Ghrelin O-Acyltransferase Regulate Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Response to Acute Stress

    NARCIS (Netherlands)

    Stark, R.; Santos, V.V.; Geenen, B.; Cabral, A.; Dinan, T.; Bayliss, J.A.; Lockie, S.H.; Reichenbach, A.; Lemus, M.B.; Perello, M.; Spencer, S.J.; Kozicz, L.T.; Andrews, Z.B.

    2016-01-01

    Ghrelin exists in two forms in circulation, acyl ghrelin and des-acyl ghrelin, both of which have distinct and fundamental roles in a variety of physiological functions. Despite this fact, a large proportion of papers simply measure and refer to plasma ghrelin without specifying the acylation

  18. Discovery of an Orally Bioavailable Benzimidazole Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor That Suppresses Body Weight Gain in Diet-Induced Obese Dogs and Postprandial Triglycerides in Humans.

    Science.gov (United States)

    Nakajima, Katsumasa; Chatelain, Ricardo; Clairmont, Kevin B; Commerford, Renee; Coppola, Gary M; Daniels, Thomas; Forster, Cornelia J; Gilmore, Thomas A; Gong, Yongjin; Jain, Monish; Kanter, Aaron; Kwak, Youngshin; Li, Jingzhou; Meyers, Charles D; Neubert, Alan D; Szklennik, Paul; Tedesco, Vivienne; Thompson, James; Truong, David; Yang, Qing; Hubbard, Brian K; Serrano-Wu, Michael H

    2017-06-08

    Modification of a gut restricted class of benzimidazole DGAT1 inhibitor 1 led to 9 with good oral bioavailability. The key structural changes to 1 include bioisosteric replacement of the amide with oxadiazole and α,α-dimethylation of the carboxylic acid, improving DGAT1 potency and gut permeability. Since DGAT1 is expressed in the small intestine, both 1 and 9 can suppress postprandial triglycerides during acute oral lipid challenges in rats and dogs. Interestingly, only 9 was found to be effective in suppressing body weight gain relative to control in a diet-induced obese dog model, suggesting the importance of systemic inhibition of DGAT1 for body weight control. 9 has advanced to clinical investigation and successfully suppressed postprandial triglycerides during an acute meal challenge in humans.

  19. 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. holarctic...a] ref|YP_762841.1| acyltransferase [Francisella tularensis subsp. holarctica OSU18] ref|YP_00142762...6.1| acyltransferase [Francisella 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. holarctic

  20. 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% ...

  1. Gclust Server: 113967 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available Sequences(49) 377 Putative lipopolysaccharide modification acyltransferase 1 9.98e-01 0.0 0.0 0.0 0.0 3...Representative annotation Putative lipopolysaccharide modification acyltransferase Number of Sequences 1 Homologs

  2. Inhibition of Acyl-CoA: cholesterol acyltransferase (ACAT), overexpression of cholesterol transporter gene, and protection of amyloid β (Aβ) oligomers-induced neuronal cell death by tricyclic pyrone molecules.

    Science.gov (United States)

    Pokhrel, Laxman; Maezawa, Izumi; Nguyen, Thi D T; Chang, Kyeong-Ok; Jin, Lee-Way; Hua, Duy H

    2012-10-25

    A major effort in Alzheimer's disease therapeutic development has targeted Aβ and downstream events. We have synthesized a small library of tricyclic pyrone compounds. Their protective action in MC65 cells and inhibition of ACAT along with the upregulation of cholesterol transporter gene were investigated. Five active compounds exhibited potencies in the nanomolar ranges. The multiple effects of the compounds on Aβ and cellular cholesterol pathways could be potential mechanisms underlying the protective effects in vivo.

  3. Androgen-mediated cholesterol metabolism in LNCaP and PC-3 cell lines is regulated through two different isoforms of acyl-coenzyme A:Cholesterol Acyltransferase (ACAT).

    Science.gov (United States)

    Locke, Jennifer A; Wasan, Kishor M; Nelson, Colleen C; Guns, Emma S; Leon, Carlos G

    2008-01-01

    The objective of this work was to determine the effect of an androgen agonist, R1881, on intracellular cholesterol synthesis and esterification in androgen-sensitive (AS) prostate cancer (LNCaP) cells. We investigated the activity and expression of cholesterol metabolism enzymes, HMG-CoA-reductase and ACAT in the LNCaP and PC-3 (androgen-independent control) models. Microsomal PC-3 HMG-CoA-reductase activity was increased with R1881 despite having similar cholesterol levels while increased cholesterol levels in microsomes from LNCaPs treated with R1881 (L+) were associated with increased HMG-CoA reductase activity. Increased intracellular cholesteryl esters (CE) found in (L+) were not associated with an increased ACAT1 activity. There was no effect from androgen treatment on ACAT1 protein expression in theses cells; however, ACAT2 expression was induced upon R1881 treatment. In contrast, we found an increase in the in vitro ACAT1 activity in PC-3 cells treated with androgen (P+). Only ACAT1 expression was induced in P+. We further assessed the expression of STAT1 alpha, a transcriptional activator that modulates ACAT1 expression. STAT1 alpha expression and phosphorylation were induced in P+. To determine the role of the AR on ACAT1 expression and esterification, we treated PC-3 cells overexpressing the androgen receptor with R1881 (PAR+). AR expression was decreased in PAR+ cells; ACAT1 protein expression and cholesterol ester levels were also decreased, however, ACAT2 remained unchanged. STAT1 alpha expression was decreased in PAR+. Overall, these findings support the importance of cholesterol metabolism regulation within prostate cancer cells and unravel a novel role for STAT1 alpha in prostate cancer metabolism. (c) 2007 Wiley-Liss, Inc.

  4. Plasma levels of lecithin:cholesterol acyltransferase and risk of future coronary artery disease in apparently healthy men and women : a prospective case-control analysis nested in the EPIC-Norfolk population study

    NARCIS (Netherlands)

    Holleboom, A G; Kuivenhoven, J A; Vergeer, M; Hovingh, G K; van Miert, J N; Wareham, N J; Kastelein, J J P; Khaw, K-T; Boekholdt, S M

    LCAT plays a key role in the maturation of HDL, as evidenced by low HDL-cholesterol levels in carriers of deleterious mutations in LCAT. However, the role of LCAT in atherosclerosis is unclear. We set out to study this in a prospective study. Plasma LCAT levels, which strongly correlate with LCAT

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|YP_011077.1| apolipoprotein N-acyltransferase [Desulfovibrio ...vulgaris subsp. vulgaris str. Hildenborough] ref|YP_966749.1| apolipoprotein N-acyltransferase [Desulfovibri...o vulgaris subsp. vulgaris DP4] gb|AAS96336.1| apolipoprotein N-acyltransferase [Desulfovibrio vulgaris subs...p. vulgaris str. Hildenborough] gb|ABM28322.1| apolipoprotein N-acyltransferase [Desulfovibrio vulgaris subsp. vulgaris DP4] YP_011077.1 2e-26 34% ...

  6. Protein (Cyanobacteria): 427702040 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 2 292564:682 ... UDP-3-O-(3-hydroxymyristoyl) glucosamine N-acyltransferase Cyanobium gracile PCC 6307 MRFSSLLTRIGA...ATGWVKMPQTGRVVLEDGVEVGCGSTIDRPSVGETRIGAGTKIDNLVHVGHGVVTGKGCALAAQVGIAGGAVLGNGVILAGQVGVANRAVIGDRAIASSKSGIHGEIAAGEVVSGYPAIPNRLWLRCSAVFNKLPEMARTLRQLQK

  7. 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 avena...e subsp. citrulli AAC00-1] gb|ABM34725.1| apolipoprotein N-acyltransferase [Acidovorax avenae subsp. citrulli AAC00-1] YP_972499.1 1e-177 68% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|ZP_01579180.1| apolipoprotein N-acyltransferase [Delftia acid...ovorans SPH-1] gb|EAV76116.1| apolipoprotein N-acyltransferase [Delftia acidovorans SPH-1] ZP_01579180.1 1e-147 60% ...

  9. 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% ...

  10. 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% ...

  11. 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

  12. 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% ...

  13. 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. syri...ngae B728a] gb|AAY40114.1| Acyltransferase 3 [Pseudomonas syringae pv. syringae B728a] YP_238152.1 1e-75 48% ...

  14. 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

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|YP_385317.1| apolipoprotein N-acyltransferase [Geobacter metallireduce...ns GS-15] gb|ABB32592.1| Apolipoprotein N-acyltransferase [Geobacter metallireducens GS-15] YP_385317.1 2e-24 31% ...

  16. 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 thai...landensis E264] gb|ABC38658.1| acyltransferase family protein [Burkholderia thailandensis E264] YP_442494.1 2e-52 44% ...

  17. AcEST: BP920682 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ICK Membrane-bound O-acyltransferase domain-containing protein 2 OS=Gallus gallus GN=mboat2 PE=2 SV=1 Length...182 >sp|Q3T1J2|MBOA2_RAT Membrane-bound O-acyltransferase domain-containing protein 2 OS=Rattus norvegicus GN=Mboat

  18. 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 [Alkalilimnicol...a ehrlichei MLHE-1] gb|ABI55752.1| apolipoprotein N-acyltransferase [Alkalilimnicola ehrlichei MLHE-1] YP_741242.1 7e-05 33% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|ZP_01014652.1| apolipoprotein N-acyltransferase [Rhodobactera...les bacterium HTCC2654] gb|EAQ11675.1| apolipoprotein N-acyltransferase [Rhodobacterales bacterium HTCC2654] ZP_01014652.1 4e-31 31% ...

  20. NCBI nr-aa BLAST: CBRC-MDOM-04-0080 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-04-0080 ref|ZP_05081345.1| apolipoprotein N-acyltransferase [beta proteob...acterium KB13] gb|EDZ64032.1| apolipoprotein N-acyltransferase [beta proteobacterium KB13] ZP_05081345.1 0.052 24% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-MLUC-01-0475 ref|YP_002552020.1| apolipoprotein N-acyltransferase [Diaphorobac...ter sp. TPSY] gb|ACM32020.1| apolipoprotein N-acyltransferase [Diaphorobacter sp. TPSY] YP_002552020.1 0.24 25% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|ZP_00964341.1| apolipoprotein N-acyltransferase [Sulfitobacte...r sp. NAS-14.1] gb|EAP78981.1| apolipoprotein N-acyltransferase [Sulfitobacter sp. NAS-14.1] ZP_00964341.1 3e-26 31% ...

  3. 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% ...

  4. 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% ...

  5. NCBI nr-aa BLAST: CBRC-XTRO-01-0090 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0090 ref|ZP_01469603.1| possible apolipoprotein n-acyltransferase [Syn...echococcus sp. BL107] gb|EAU70617.1| possible apolipoprotein n-acyltransferase [Synechococcus sp. BL107] ZP_01469603.1 0.025 27% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_524928.1| apolipoprotein N-acyltransferase [Rhodoferax fer...rireducens T118] gb|ABD71397.1| apolipoprotein N-acyltransferase [Rhodoferax ferrireducens DSM 15236] YP_524928.1 1e-143 56% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|YP_844856.1| apolipoprotein N-acyltransferase [Syntrophobacte...r fumaroxidans MPOB] gb|ABK16421.1| apolipoprotein N-acyltransferase [Syntrophobacter fumaroxidans MPOB] YP_844856.1 2e-24 33% ...

  8. NCBI nr-aa BLAST: CBRC-TTRU-01-0349 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0349 ref|ZP_03046466.1| AatD, apolipoprotein N-acyltransferase [Escher...ichia coli E22] gb|EDV81547.1| AatD, apolipoprotein N-acyltransferase [Escherichia coli E22] ZP_03046466.1 0.065 24% ...

  9. 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| apo...lipoprotein N-acyltransferase [Acidovorax sp. JS42] YP_984850.1 0.0 70% ...

  10. NCBI nr-aa BLAST: CBRC-OLAT-13-0071 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OLAT-13-0071 ref|YP_001072768.1| apolipoprotein N-acyltransferase [Mycobacteri...um sp. JLS] gb|ABO00278.1| apolipoprotein N-acyltransferase [Mycobacterium sp. JLS] YP_001072768.1 0.11 31% ...

  11. 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% ...

  12. NCBI nr-aa BLAST: CBRC-XTRO-01-1006 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-1006 ref|YP_461586.1| apolipoprotein N-acyltransferase [Syntrophus aci...ditrophicus SB] gb|ABC77418.1| apolipoprotein N-acyltransferase [Syntrophus aciditrophicus SB] YP_461586.1 6.7 25% ...

  13. NCBI nr-aa BLAST: CBRC-LAFR-01-2722 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-2722 ref|YP_996924.1| apolipoprotein N-acyltransferase [Verminephrobac...ter eiseniae EF01-2] gb|ABM57906.1| apolipoprotein N-acyltransferase [Verminephrobacter eiseniae EF01-2] YP_996924.1 1.3 29% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0138 ref|YP_865356.1| apolipoprotein N-acyltransferase [Magnetococcus ...sp. MC-1] gb|ABK43950.1| apolipoprotein N-acyltransferase [Magnetococcus sp. MC-1] YP_865356.1 1e-178 100% ...

  15. NCBI nr-aa BLAST: CBRC-GACU-09-0012 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-09-0012 ref|ZP_01737953.1| apolipoprotein N-acyltransferase [Marinobacter... sp. ELB17] gb|EAZ99314.1| apolipoprotein N-acyltransferase [Marinobacter sp. ELB17] ZP_01737953.1 4.9 37% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0530 ref|YP_996924.1| apolipoprotein N-acyltransferase [Verminephrobac...ter eiseniae EF01-2] gb|ABM57906.1| apolipoprotein N-acyltransferase [Verminephrobacter eiseniae EF01-2] YP_996924.1 0.0 73% ...

  17. NCBI nr-aa BLAST: CBRC-RNOR-03-0515 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-RNOR-03-0515 ref|NP_711305.1| apolipoprotein N-acyltransferase [Leptospira int...ase 1) gb|AAN48323.1|AE011295_2 apolipoprotein N-acyltransferase [Leptospira interrogans serovar Lai str. 56601] NP_711305.1 0.31 22% ...

  18. NCBI nr-aa BLAST: CBRC-XTRO-01-3405 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3405 ref|ZP_01469603.1| possible apolipoprotein n-acyltransferase [Syn...echococcus sp. BL107] gb|EAU70617.1| possible apolipoprotein n-acyltransferase [Synechococcus sp. BL107] ZP_01469603.1 0.007 29% ...

  19. 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% ...

  20. Gclust Server: 91331 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 303 Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) 1 1.00e-40 0.0 0.0 4.0...annotation Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) Number of Sequences 1 Homologs

  1. Gclust Server: 95189 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 319 Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) 1 1.00e-25 0.0 0.0 4.0...annotation Predicted hydrolases or acyltransferases (alpha/beta hydrolase superfamily) Number of Sequences 1 Homologs

  2. NCBI nr-aa BLAST: CBRC-TGUT-18-0003 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-18-0003 ref|YP_880644.1| putative acyltransferase, putative [Mycobacterium avium... 104] gb|ABK66665.1| putative acyltransferase, putative [Mycobacterium avium 104] YP_880644.1 6.4 31% ...

  3. 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% ...

  4. 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% ...

  5. 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 bauman...nii ATCC 17978] gb|ABO13799.1| putative acyltransferase [Acinetobacter baumannii ATCC 17978] YP_001086401.1 1e-67 70% ...

  6. SwissProt search result: AK119872 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119872 002-179-D03 (Q9ES71) Dihydroxyacetone phosphate acyltransferase (EC 2.3.1....42) (DHAP-AT) (DAP-AT) (Glycerone-phosphate O-acyltransferase) (Acyl-CoA:dihydroxyacetonephosphateacyltransferase) GNPAT_RAT 1e-55 ...

  7. SwissProt search result: AK119872 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119872 002-179-D03 (P98192) Dihydroxyacetone phosphate acyltransferase (EC 2.3.1....42) (DHAP-AT) (DAP-AT) (Glycerone-phosphate O-acyltransferase) (Acyl-CoA:dihydroxyacetonephosphateacyltransferase) GNPAT_MOUSE 8e-56 ...

  8. SwissProt search result: AK119872 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119872 002-179-D03 (O15228) Dihydroxyacetone phosphate acyltransferase (EC 2.3.1....42) (DHAP-AT) (DAP-AT) (Glycerone-phosphate O-acyltransferase) (Acyl-CoA:dihydroxyacetonephosphateacyltransferase) GNPAT_HUMAN 9e-55 ...

  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. ORF Alignment: NC_000913 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... adenylate kinase activity; pleiotropic effects on ... glycerol-3-phosphate acyltransferase...ylate kinase activity; ... pleiotropic effects on glycerol-3-phosphate ... acyltransferase act...TP-AMP ... transphosphorylase) (AK) ref|NP_286215.1| adenylate ... kinase activity; pleiotropic effects

  11. ORF Alignment: NC_002655 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... adenylate kinase activity; pleiotropic effects on ... glycerol-3-phosphate acyltransferase...ylate kinase activity; ... pleiotropic effects on glycerol-3-phosphate ... acyltransferase act...TP-AMP ... transphosphorylase) (AK) ref|NP_286215.1| adenylate ... kinase activity; pleiotropic effects

  12. ORF Alignment: NC_002695 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ... adenylate kinase activity; pleiotropic effects on ... glycerol-3-phosphate acyltransferase...ylate kinase activity; ... pleiotropic effects on glycerol-3-phosphate ... acyltransferase act...TP-AMP ... transphosphorylase) (AK) ref|NP_286215.1| adenylate ... kinase activity; pleiotropic effects

  13. 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% ...

  14. 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% ...

  15. NCBI nr-aa BLAST: CBRC-LAFR-01-0356 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-0356 ref|YP_266901.1| glycerol-3-phosphate acyltransferase [Colwellia ...psychrerythraea 34H] gb|AAZ27641.1| glycerol-3-phosphate acyltransferase [Colwellia psychrerythraea 34H] YP_266901.1 0.96 34% ...

  16. NCBI nr-aa BLAST: CBRC-MDOM-07-0000 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-07-0000 ref|ZP_05664628.1| acyltransferase [Enterococcus faecium 1,231,50...1] gb|EEV47961.1| acyltransferase [Enterococcus faecium 1,231,501] ZP_05664628.1 0.019 23% ...

  17. 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% ...

  18. 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% ...

  19. 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% ...

  20. NCBI nr-aa BLAST: CBRC-TTRU-01-0101 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0101 ref|YP_001833721.1| acyltransferase 3 [Beijerinckia indica subsp. indica... ATCC 9039] gb|ACB96232.1| acyltransferase 3 [Beijerinckia indica subsp. indica ATCC 9039] YP_001833721.1 0.43 27% ...

  1. NCBI nr-aa BLAST: CBRC-TTRU-01-0528 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0528 ref|YP_001833721.1| acyltransferase 3 [Beijerinckia indica subsp. indica... ATCC 9039] gb|ACB96232.1| acyltransferase 3 [Beijerinckia indica subsp. indica ATCC 9039] YP_001833721.1 2e-04 32% ...

  2. AcEST: BP917830 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Q5ZKL6|MBOA2_CHICK Membrane-bound O-acyltransferase domain-containing protein 2 OS=Gallus gallus GN=mboat2 P...-acyltransferase domain-containing protein 2 OS=Rattus norvegicus GN=Mboat2 PE=2 SV=2 Length = 519 Score = 6...sferase domain-containing protein 2 OS=Mus musculus GN=Mboat2 PE=2 SV=1 Length = ...ne-bound O-acyltransferase domain-containing protein 1 OS=Mus musculus GN=Mboat1 PE=2 SV=1 Length = 492 Scor

  3. ACAT Inhibition and Progression of Carotid Atherosclerosis in Patients With Familial Hypercholesterolemia The CAPTIVATE Randomized Trial

    NARCIS (Netherlands)

    Meuwese, Marijn C.; de Groot, Eric; Duivenvoorden, Raphaël; Trip, Mieke D.; Ose, Leiv; Maritz, Frans J.; Basart, Dick C. G.; Kastelein, John J. P.; Habib, Rafik; Davidson, Michael H.; Zwinderman, Aeilko H.; Schwocho, Lee R.; Stein, Evan A.

    2009-01-01

    Context Inhibition of acyl coenzyme A: cholesterol acyltransferase (ACAT), an intracellular enzyme involved in cholesterol accumulation, with pactimibe was developed to assist in the prevention of cardiovascular disease. Objective To evaluate the efficacy and safety of pactimibe in inhibition of

  4. NCBI nr-aa BLAST: CBRC-PVAM-01-1348 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PVAM-01-1348 ref|NP_060309.2| lysophosphatidylcholine acyltransferase 2 [Homo ...erase 2; Short=LPCAT-2; AltName: Full=1-alkylglycerophosphocholine O-acetyltransferase; AltName: Full=Acetyl

  5. Presidential Green Chemistry Challenge: 2012 Greener Synthetic Pathways Award

    Science.gov (United States)

    Presidential Green Chemistry Challenge 2012 award winner, Codexis and Professor Yi Tang, developed a synthesis for the high cholesterol drug, simvastatin, using an engineered acyltransferase enzyme and a low-cost acyl donor as a feedstock.

  6. 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

  7. 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

  8. 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

  9. The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii*

    Science.gov (United States)

    Serebryakova, Marina V.; Demina, Irina A.; Galyamina, Maria A.; Kondratov, Ilya G.; Ladygina, Valentina G.; Govorun, Vadim M.

    2011-01-01

    Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria. PMID:21540185

  10. Gclust Server: 99168 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 99168 Mes_Meso_3004 Cluster Sequences Related Sequences(179) 283 Nitrilase/cyanide hydratase... and apolipoprotein N-acyltransferase 1 1.00e-19 0.0 0.0 0.0 0.0 3.23 0.0 Show 99168 Cluster ID 99168 Se...quence ID Mes_Meso_3004 Link to cluster sequences Cluster Sequences Link to related sequences Related Sequences(179) Se... and apolipoprotein N-acyltransferase Number of Sequences 1 Homologs 1 Clustering threshold 1.00e-19 P...quence length 283 Representative annotation Nitrilase/cyanide hydratase

  11. Gclust Server: 100233 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 100233 Rpa4_RPB_0024 Cluster Sequences Related Sequences(170) 292 Nitrilase/cyanide hydratase... and apolipoprotein N-acyltransferase 1 9.98e-01 0.0 0.0 0.0 6.67 0.0 0.0 Show 100233 Cluster ID 100233 Se...quence ID Rpa4_RPB_0024 Link to cluster sequences Cluster Sequences Link to related sequences Related Sequences(170) Se... and apolipoprotein N-acyltransferase Number of Sequences 1 Homologs 1 Clustering threshold 9.98e-0...quence length 292 Representative annotation Nitrilase/cyanide hydratase

  12. Effect of DGAT1 gene mutation in sows of dam-line on the ...

    African Journals Online (AJOL)

    Diacylglycerol acyltransferase 1 gene (DGAT1) involved in the synthesis and transport of triglycerides is located on chromosome 4 in pigs, in the region with about 200 QTLs responsible among other things for: fat thickness, daily gains, fat content and composition of fatty acids. It is thus probable that the gene polymorphism ...

  13. AcEST: BP913884 [AcEST

    Lifescience Database Archive (English)

    Full Text Available oprotein N-acyltransferase OS=Neisseri... 33 0.61 sp|Q91610|DHH1_XENLA Desert hedgehog protein 1 OS=Xenopus ...VQTN 460 +HM+TL+ + WL++ N Sbjct: 168 LGGIHMVTLATAFLGVWLVLASNN 191 >sp|Q91610|DHH1_XENLA Desert

  14. And then there were acyl coenzyme A:cholesterol acyl transferase inhibitors

    NARCIS (Netherlands)

    Meuwese, Marijn C.; Franssen, Remco; Stroes, Erik S. G.; Kastelein, John J. P.

    2006-01-01

    PURPOSE OF REVIEW: The reputation of acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitors has changed profoundly from promising new drugs for cardiovascular prevention to drugs without clinical benefits or possibly even with adverse effects. RECENT FINDINGS: ACAT inhibitors decrease the

  15. 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% ...

  16. Unconjugated bile salts shuttle through hepatocyte peroxisomes for taurine conjugation.

    NARCIS (Netherlands)

    Rembacz, K.P.; Woudenberg, J.; Hoekstra, M.; Jonkers, E.Z.; Heuvel, F.A. van den; Buist-Homan, M.; Woudenberg-Vrenken, T.E.; Rohacova, J.; Marin, M.L.; Miranda, M.A.; Moshage, H.; Stellaard, F.; Faber, K.N.

    2010-01-01

    Bile acid-CoA:amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein,

  17. 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% ...

  18. 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

  19. Olea europaea Linn (Oleaceae) Fruit Pulp Exhibits ...

    African Journals Online (AJOL)

    as hydroxymethylglutaryl-CoA reductase (HMGR), acyl-CoA:cholesterol acyltransferase (ACAT), cytochrome P450 7A1 (CYP7A1) and peroxisome proliferation-activated receptor alpha (PPAR-á) in rat livers were evaluated using Western blotting. Results: OFP-EA-extract markedly altered the increased plasma TC, TG, LDL ...

  20. Unconjugated Bile Salts Shuttle Through Hepatocyte Peroxisomes for Taurine Conjugation

    NARCIS (Netherlands)

    Rembacz, Krzysztof P.; Woudenberg, Jannes; Hoekstra, Mark; Jonkers, Elles Z.; van den Heuvel, Fiona A. J.; Buist-Homan, Manon; Woudenberg-Vrenken, Titia E.; Rohacova, Jana; Luisa Marin, M.; Miranda, Miguel A.; Moshage, Han; Stellaard, Frans; Faber, Klaas Nico

    2010-01-01

    Bile acid-CoA.amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation Recently, we showed that BAAT is a peroxisomal protein,

  1. Reference: 42 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available et al. 2003 Aug. Plant Cell 15(8):1872-87. Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the init...ial step of glycerolipid biosynthesis in the extraplastidic compartments of plant c

  2. Ectopic expression of Crambe abyssinica lysophosphatidic acid ...

    African Journals Online (AJOL)

    USER

    2010-06-21

    Jun 21, 2010 ... lysophosphatidic acid acyltransferase in transgenic rapeseed increases its oil content ... T1 generation demonstrated that they were at the similar level in both transgenic plants and their non- transgenic counterparts. .... putative LPAAT proteins of other plant species in GenBank was created by using the ...

  3. Functional characterization of enzymes forming volatile esters from strawberry and banana

    NARCIS (Netherlands)

    Beekwilder, M.J.; Alvarez-Huerta, M.; Neef, E.J.; Verstappen, F.W.A.; Bouwmeester, H.J.; Aharoni, A.

    2004-01-01

    Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and

  4. GenBank blastx search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 BC089821.1 Rattus norvegicus acetyl-Coenzyme A acyltransferase... 1 (peroxisomal 3-oxoacyl-Coenzyme A thiolase), mRNA (cDNA clone MGC:108766 IMAGE:7122340), complete cds.|ROD ROD 6e-70 +3 ...

  5. GenBank blastx search result: AK061948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061948 001-042-E07 BC011977.1 Homo sapiens acetyl-Coenzyme A acyltransferase 1 (peroxisomal 3-oxoacyl-Coen...zyme A thiolase), mRNA (cDNA clone MGC:9371 IMAGE:3860150), complete cds.|PRI PRI 1e-104 +3 ...

  6. GenBank blastx search result: AK059654 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059654 001-031-C10 BC011977.1 Homo sapiens acetyl-Coenzyme A acyltransferase 1 (peroxisomal 3-oxoacyl-Coen...zyme A thiolase), mRNA (cDNA clone MGC:9371 IMAGE:3860150), complete cds.|PRI PRI 2e-53 +1 ...

  7. Peroxisomes are required for efficient penicillin biosynthesis in Penicillium chrysogenum

    NARCIS (Netherlands)

    Meijer, Wiebe H.; Gidijala, Loknath; Fekken, Susan; Kiel, Jan A. K. W.; van den Berg, Marco A.; Lascaris, Romeo; Bovenberg, Roel A. L.; van der Klei, Ida J.

    In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the

  8. A prenatal test for the cerebro-hepato-renal (Zellweger) syndrome by demonstration of the absence of catalase-containing particles (peroxisomes) in cultured amniotic fluid cells

    NARCIS (Netherlands)

    Wanders, R. J.; Schrakamp, G.; van den Bosch, H.; Tager, J. M.; Schutgens, R. B.

    1986-01-01

    In this paper we show that whereas acyl-CoA: dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient in homogenates of cultured amniotic fluid cells of fetuses with Zellweger syndrome, catalase a soluble peroxisomal matrix enzyme is present in normal amounts.

  9. 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.

  10. Revisiting the gram-negative lipoprotein paradigm

    Science.gov (United States)

    The processing of lipoproteins (lpps) in Gram-negative bacteria is generally considered to be an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein n-acyltransferase. The mature lipoproteins are then sorted...

  11. Effects of the DGAT1 polymorphism on test-day milk production traits throughout lactation

    NARCIS (Netherlands)

    Bovenhuis, H.; Visker, M.H.P.W.; Valenberg, van H.J.F.; Buitenhuis, A.J.; Arendonk, van J.A.M.

    2015-01-01

    Several studies have shown that the diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism has a major effect on milk production traits. It is less clear how effects of DGAT1 on milk production traits change throughout lactation, if dominance effects of DGAT1 are relevant, and whether DGAT1

  12. 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...

  13. 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...

  14. 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...

  15. 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

  16. Heterologous expression of two GPATs from Jatropha curcas alters seed oil levels in transgenic Arabidopsis thaliana.

    Science.gov (United States)

    Misra, Aparna; Khan, Kasim; Niranjan, Abhishek; Kumar, Vinod; Sane, Vidhu A

    2017-10-01

    Oils and fats are stored in endosperm during seed development in the form of triacylglycerols. Three acyltransferases: glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidyl acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) are involved in the storage lipid biosynthesis and catalyze the stepwise acylation of glycerol backbone. In this study two members of GPAT gene family (JcGPAT1 and JcGPAT2) from Jatropha seeds were identified and characterized. Sequence analysis suggested that JcGPAT1 and JcGPAT2 are homologous to Arabidopsis acyltransferase-1 (ATS1) and AtGPAT9 respectively. The sub-cellular localization studies of these two GPATs showed that JcGPAT1 localizes into plastid whereas JcGPAT2 localizes in to endoplasmic reticulum. JcGPAT1 and JcGPAT2 expressed throughout the seed development with higher expression in fully matured seed compared to immature seed. The transcript levels of JcGPAT2 were higher in comparison to JcGPAT1 in different developmental stages of seed. Over-expression of JcGPAT1 and JcGPAT2 under constitutive and seed specific promoters in Arabidopsis thaliana increased total oil content. Transgenic seeds of JcGPAT2-OE lines accumulated 43-60% more oil than control seeds whereas seeds of Arabidopsis lines over-expressing plastidial GPAT lead to only 13-20% increase in oil content. Functional characterization of GPAT homologues of Jatropha in Arabidopsis suggested that these are involved in oil biosynthesis but might have specific roles in Jatropha. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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.

  18. Tissue-specific knockouts of ACAT2 reveal that intestinal depletion is sufficient to prevent diet-induced cholesterol accumulation in the liver and blood[S

    OpenAIRE

    Zhang, Jun; Kelley, Kathryn L.; Marshall, Stephanie M.; Davis, Matthew A.; Wilson, Martha D.; Sawyer, Janet K.; Farese, Robert V.; Brown, J. Mark; Rudel, Lawrence L.

    2012-01-01

    Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol hom...

  19. ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane

    OpenAIRE

    Joyce, Charles W.; Shelness, Gregory S.; Davis, Matthew A.; Lee, Richard G.; Skinner, Kelly; Anderson, Richard A.; Rudel, Lawrence L.

    2000-01-01

    A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determi...

  20. Cell wall composition and candidate biosynthesis gene expression during rice development

    DEFF Research Database (Denmark)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra

    2016-01-01

    , we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had...... strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically...

  1. Disorders of Lipid Metabolism and its Correction in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    O.O. Melnyk

    2016-04-01

    Full Text Available Chronic kidney disease — a proven risk factor of the development and progression of lipid metabolism disorders. The basis of these disorders — an increase in blood plasma cholesterol, triglycerides, low density lipoproteins and decreased levels of high density lipoproteins, apo AI and apo AII. There has been a decrease in the activity of enzymes: lipoprotein lipase, hepatic triglyceride lipase, lecithin-cholesterol acyltransferase. The use of lipid-modifying drugs — statins, fibrates, nicotinic acid was proposed.

  2. 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.

  3. Sudden death due to paralysis and synaptic and behavioral deficits when Hip14/Zdhhc17 is deleted in adult mice

    OpenAIRE

    Sanders, Shaun S.; Parsons, Matthew P.; Katherine K N Mui; Southwell, Amber L.; Franciosi, Sonia; Cheung, Daphne; Waltl, Sabine; Raymond, Lynn A.; Hayden, Michael R.

    2016-01-01

    Background Palmitoylation, the addition of palmitate to proteins by palmitoyl acyltransferases (PATs), is an important regulator of synaptic protein localization and function. Many palmitoylated proteins and PATs have been implicated in neuropsychiatric diseases, including Huntington disease, schizophrenia, amyotrophic lateral sclerosis, Alzheimer disease, and X-linked intellectual disability. HIP14/DHHC17 is the most conserved PAT that palmitoylates many synaptic proteins. Hip14 hypomorphic ...

  4. Drugs affecting the synthesis of glycerides and phospholipids in rat liver. The effects of clofibrate, halofenate, fenfluramine, amphetamine, cinchocaine, chlorpromazine, demethylimipramine, mepyramine and some of their derivatives.

    Science.gov (United States)

    Brindley, D N; Bowley, M

    1975-01-01

    The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered. PMID:1200988

  5. 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.

  6. The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum.

    OpenAIRE

    Broadway, Neil M; Pease, Richard J.; Birdsey, Graeme; Shayeghi, Majid; Turner, Nigel A; David Saggerson, E

    2003-01-01

    Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 54...

  7. Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

    OpenAIRE

    Nishi, Yoshihiro; Mifune, Hiroharu; Yabuki, Akira; Tajiri, Yuji; Hirata, Rumiko; Tanaka, Eiichiro; Hosoda, Hiroshi; Kangawa, Kenji; Kojima, Masayasu

    2013-01-01

    Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the sub-cellular processes governing the acylation of ghrelin remain to be elucidated.Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-pro...

  8. Diacylglycerol acyl transferase: A pathogenicity related gene in Colletotrichum gloeosporioides.

    Science.gov (United States)

    Sharma, Meenakshi; Guleria, Shiwani; Kulshrestha, Saurabh

    2016-11-01

    To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, restriction enzyme-mediated integration (REMI) mutagenesis identified the mutants of C. gloeosporioides impaired in pathogenicity. Transformants screened for defects in pathogenicity using detached leaves and fruits. Of the 20 REMI transformants tested, two mutants (H4 and H7) showed reduced pathogenicity on leaves of apple, kiwi, mango, peach, and fruits of guava, apple, and capsicum. One tagged gene from the genome sequence of mutant H4 was recovered by inverse PCR. Sequence analysis of the tagged site in mutant H4 revealed insertion in diacylglycerol acyltransferase gene which encodes diacylglycerol acyltransferase enzyme, catalyzing the steps involved in the biosynthesis of triacylglycerol, an important component of biological membranes and source of energy. Therefore, tagging of diacylglycerol acyltransferase gene in mutant H4 resulted in reduced pathogenicity, indicating possible role of this gene in pathogenicity of C. gloeosporioides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro.

    Science.gov (United States)

    Nancolas, Bethany; Bull, Ian D; Stenner, Richard; Dufour, Virginie; Curnow, Paul

    2017-06-01

    The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

  10. 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.

  11. ACAT-selective and nonselective DGAT1 inhibition: adrenocortical effects--a cross-species comparison.

    Science.gov (United States)

    Floettmann, Jan Eike; Buckett, Linda K; Turnbull, Andrew V; Smith, Tim; Hallberg, Carina; Birch, Alan; Lees, David; Jones, Huw B

    2013-01-01

    Acyl-coenzyme A: cholesterol O-Acyltransferase (ACAT) and Acyl-coenzyme A: diacylglycerol O-acyltransferase (DGAT) enzymes play important roles in synthesizing neutral lipids, and inhibitors of these enzymes have been investigated as potential treatments for diabetes and other metabolic diseases. Administration of a Acyl-coenzyme A: diacylglycerol O-acyltransferase 1 (DGAT1) inhibitor with very limited cellular selectivity over ACAT resulted in significant adrenocortical degenerative changes in dogs. These changes included macrosteatotic vacuolation associated with adrenocyte cell death in the zonae glomerulosa and fasciculata and minimal to substantial mixed inflammatory cell infiltration and were similar to those described previously for some ACAT inhibitors in dogs. In the mouse, similar but only transient adrenocortical degenerative changes were seen as well as a distinctive nondegenerative reduction in cortical fine vacuolation. In the marmoset, only the distinctive nondegenerative reduction in cortical fine vacuolation was observed, suggesting that the dog, followed by the mouse, is the most sensitive species for cortical degeneration. Biochemical analysis of adrenal cholesterol and cholesteryl ester indicated that the distinctive reduction in cortical fine vacuolation correlated with a significant reduction in cholesteryl ester in the mouse and marmoset, whereas no significant reduction in cholestryl ester, but an increase in free cholesterol was observed in dogs. Administration of a DGAT1 inhibitor with markedly improved selectivity over ACAT to the marmoset and the mouse resulted in no adrenal pathology at exposures sufficient to cause substantial DGAT1 but not ACAT inhibition, thereby implicating ACAT rather than DGAT1 inhibition as the probable cause of the observed adrenal changes. Recognizing that the distinctive nondegenerative reduction in cortical fine vacuolation in the mouse could be used as a histopathological biomarker for an in vivo model of

  12. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds.

    Science.gov (United States)

    Durrett, Timothy P; McClosky, Daniel D; Tumaney, Ajay W; Elzinga, Dezi A; Ohlrogge, John; Pollard, Mike

    2010-05-18

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

  13. Functional characterization of enzymes forming volatile esters from strawberry and banana.

    Science.gov (United States)

    Beekwilder, Jules; Alvarez-Huerta, Mayte; Neef, Evert; Verstappen, Francel W A; Bouwmeester, Harro J; Aharoni, Asaph

    2004-08-01

    Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria x ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants.

  14. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity.

    Science.gov (United States)

    Saerens, Sofie M G; Verstrepen, Kevin J; Van Laere, Stijn D M; Voet, Arnout R D; Van Dijck, Patrick; Delvaux, Freddy R; Thevelein, Johan M

    2006-02-17

    Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a alpha/beta-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.

  15. A regulatory role of LPCAT1 in the synthesis of inflammatory lipids, PAF and LPC, in the retina of diabetic mice.

    Science.gov (United States)

    Cheng, Long; Han, Xiao; Shi, Yuguang

    2009-12-01

    Platelet-activating factor (PAF) and lysophosphatidylcholine (LPC) are potent inflammatory lipids. Elevated levels of PAF and LPC are associated with the onset of diabetic retinopathy and neurodegeneration. However, the molecular mechanisms underlying such defects remain elusive. LPCAT1 is a newly reported lysophospholipid acyltransferase implicated in the anti-inflammatory response by its role in conversion of LPC to PC. Intriguingly, the LPCAT1 enzyme also catalyzes the synthesis of PAF from lyso-PAF with use of acetyl-CoA as a substrate. The present studies investigated regulatory roles of LPCAT1 in the synthesis of inflammatory lipids during the onset of diabetes. Our work shows that LPCAT1 plays an important role in the inactivation of PAF by catalyzing the synthesis of alkyl-PC, an inactivated form of PAF with use of acyl-CoA and lyso-PAF as substrates. In support of a role of LPCAT1 in anti-inflammatory responses in diabetic retinopathy, LPCAT1 is most abundantly expressed in the retina. Moreover, LPCAT1 mRNA levels and acyltransferase activity toward lyso-PAF and LPC were significantly downregulated in retina and brain tissues in response to the onset of diabetes in Ins2(Akita) and db/db mice, mouse models of type 1 and type 2 diabetes, respectively. Conversely, treatment of db/db mice with rosiglitazone, an antidiabetes compound, significantly upregulated LPCAT1 mRNA levels concurrently with increased acyltransferase activity in the retina and brain. Collectively, these findings identified a novel regulatory role of LPCAT1 in catalyzing the inactivation of inflammatory lipids in the retina of diabetic mice.

  16. 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.

  17. 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

  18. EST Table: FS881515 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS881515 E_FL_ftes_21J14_F_0 10/09/28 65 %/163 aa ref|XP_966553.1| PREDICTED: simil...] gb|EFA04227.1| hypothetical protein TcasGA2_TC014478 [Tribolium castaneum] 10/09/11 60 %/154 aa FBpp0140116|DereGG215...70-PA 10/08/29 53 %/152 aa M05B5.4#CE06190#WBGene00010872#phosphatidyl...choline-sterol acyltransferase#status:Confirmed#UniProt:Q21515#protein_ id:CAA95833.1 10/09/10 59 %/147 aa A

  19. Synthesis, biological evaluation, and molecular docking studies of xanthone sulfonamides as ACAT inhibitors.

    Science.gov (United States)

    Li, Xiang; Zou, Yan; Zhao, Qingjie; Yang, Yan; Wu, Maocheng; Huang, Ting; Hu, Honggang; Wu, Qiuye

    2015-03-01

    Three series of xanthone sulfonamides were synthesized, and their inhibitory activities against acyl-CoA: cholesterol acyltransferase (ACAT) were evaluated. Results showed that most of the title compounds exhibited strong inhibitory activity against ACAT, of which compounds 1c, 1e, 1f, 2d, 2e, and 3d were proved to be more active than the positive control Sandoz 58-035. Computational docking experiments indicated that the interaction between inhibitors and ACAT contained the H-bond interaction, the hydrophobic interaction, and the narrow hydrophobic cleft. © 2014 John Wiley & Sons A/S.

  20. Human ACAT-1 and ACAT-2 inhibitory activities of pentacyclic triterpenes from the leaves of Lycopus lucidus TURCZ.

    Science.gov (United States)

    Lee, Woo Song; Im, Kyung-Ran; Park, Yong-Dae; Sung, Nack-Do; Jeong, Tae-Sook

    2006-02-01

    Acyl-CoA: cholesterol acyltransferase (ACAT) catalyzes the acylation of cholesterol to cholesteryl ester with long chain fatty acids and ACAT inhibition is a useful strategy for treating hypercholesterolemia or atherosclerosis. Pentacyclic triterpenes, ursolic acid (1), oleanolic acid (2), and betulinic acid (3) were isolated from the methanol extracts of the leaves of Lycopus lucidus TURCZ. by bioassay-guided fractionation. The structures of compounds 1-3 were elucidated by their spectroscopic data analysis. Among them, betulinic acid (3) exhibited more potent human ACAT-1 and ACAT-2 inhibitory activities with IC(50) values of 16.2+/-0.6 and 28.8+/-1.3 microM, respectively.

  1. The hypolipidemic effect of a new ACAT inhibitor, VULM 1457, in diabetic-hypercholesterolaemic rats.

    Science.gov (United States)

    Adameová, A; Kuzelová, M; Faberová, V; Svec, P

    2005-09-01

    The use of inhibitors of enzyme acyl-CoA: cholesterol acyltransferase (ACAT) seems to be a novel potential approach for a therapeutic treatment of dyslipidaemias and atherosclerosis. VULM 1457 is an ACAT inhibitor, which has expressed potent hypolipidemic and antiatherosclerotic effects in previous studies. In this study, we used streptozocin-induced diabetic rats, which were fed a fat-cholesterol diet to evaluate the affect of VULM 1457 on the atherogenic lipids levels in both plasma and liver. VULM 1457, with a slight influence on triglyceride levels, significantly reduced plasma and hepatic cholesterol concentrations (p < 0.05, p < 0.001; respectively) in the diabetic-hypercholesterolaemic rats.

  2. Regulation of triglyceride metabolism. I. Eukaryotic neutral lipid synthesis: "Many ways to skin ACAT or a DGAT".

    Science.gov (United States)

    Turkish, Aaron; Sturley, Stephen L

    2007-04-01

    Esterification of sterols, fatty acids and other alcohols into biologically inert forms conserves lipid resources for many cellular functions. Paradoxically, the accumulation of neutral lipids such as cholesteryl ester or triglyceride, is linked to several major disease pathologies. In a remarkable example of genetic expansion, there are at least eleven acyltransferase reactions that lead to neutral lipid production. In this review, we speculate that the complexity and apparent redundancy of neutral lipid synthesis may actually hasten rather than impede the development of novel, isoform-specific, therapeutic interventions for acne, type 2 diabetes, obesity, hyperlipidemia, fatty liver disease, and atherosclerosis.

  3. Putative neuroprotective actions of N-acyl-ethanolamines

    DEFF Research Database (Denmark)

    Hansen, Harald S.; Moesgaard, B.; Petersen, G.

    2002-01-01

    N-Acyl-ethanolamines (NAEs) and their precursors, N-acyl-ethanolamine phospholipids (NAPEs), are present in the mammalian brain at levels of a few hundred picomoles/gram tissue and a few nanomoles/gram tissue, respectively. NAE-containing arachidonic acid is called anandamide, and it has attracted...... and monounsaturated fatty acids. Formation of NAPE and NAE is catalyzed by an N-acyltransferase and an NAPE-hydrolyzing phospholipase D, respectively, two enzymes that have been characterized only preliminary. Interestingly, NAPEs and NAEs accumulate in the brain in response to neurodegenerative insults at a time...

  4. Expression and Purification of Recombinant Human Apolipoprotein A-II in Pichia pastoris

    OpenAIRE

    Su, Manman; Qi, Yitian; Wang, Mingxing; Chang, Weiqin; Peng, Shuang; Xu, Tianmin; Wang, Dingding

    2013-01-01

    Apolipoprotein A-II (ApoA-II) is the second most abundant protein constituent of high-density lipoprotein (HDL). The physiologic role of ApoA-II is poorly defined. ApoA-II may inhibit lecithin:cholesterol acyltransferase and cholesteryl-ester-transfer protein activities, but may increase the hepatic lipase activity. ApoA-II may also inhibit the hepatic cholesteryl uptake from HDL probably through the scavenger receptor class B type I depending pathway. Interpretation of data from transgenic a...

  5. A Genomic Association Analysis of Milk Production Traits in Chios Sheep Breed Using Microsatellite (SSR and SNP Markers

    Directory of Open Access Journals (Sweden)

    Dimitrios Chatziplis

    2013-05-01

    Full Text Available We have previously reported the possible segregation of major gene(s for milk yield and fat corrected milk (FCM in Chios sheep breed. In this study we demonstrate the association of an SNP marker, within the last exon of the Acetyl-CoA acyltransferase 2 (ACAA2 gene, with milk yield and FCM in the same breed. Moreover, we investigate the effect of this SNP marker along with other microsatellite markers. Fifteen microsatellite markers (SSR on 13 chromosomes were genotyped on 198 ewes of the Chios breed. The association analysis revealed significant association (P

  6. 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

    -type and psi1Delta cells, microsomal membranes isolated from psi1Delta cells are devoid of the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity that is present in microsomal membranes isolated from wild-type cells. Moreover, after the expression of PSI1 in transgenic psi1Delta cells, the sn-2...... 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....

  7. Isolation and function of spinach leaf β-ketoacyl-[acyl-carrier-protein] synthases

    OpenAIRE

    Shimakata, Takashi; Stumpf, Paul K.

    1982-01-01

    Crude spinach leaf extract readily forms the stearoyl derivative of acyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When β-ketoacyl-ACP synthase {3-oxoacyl-[ACP] synthase; acyl-[ACP]:malonyl-[ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another pro...

  8. Characterization of substrate preference for Slc1p and Cst26p in Saccharomyces cerevisiae using lipidomic approaches and an LPAAT activity assay.

    Directory of Open Access Journals (Sweden)

    Guanghou Shui

    Full Text Available BACKGROUND: Phosphatidic acid (PA is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT. Recent findings have substantiated the essential roles of acyltransferases in various biological functions. METHODOLOGIES/PRINCIPAL FINDINGS: We used a flow-injection-based lipidomic approach with approximately 200 multiple reaction monitoring (MRM transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography-based multi-stage MRM approach (more than 500 MRM transitions was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates. CONCLUSIONS: We found that Slc1p utilized fatty acid (FA 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0 in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for

  9. Promiscuity, impersonation and accommodation: evolution of plant specialized metabolism.

    Science.gov (United States)

    Leong, Bryan J; Last, Robert L

    2017-12-01

    Specialized metabolic enzymes and metabolite diversity evolve through a variety of mechanisms including promiscuity, changes in substrate specificity, modifications of gene expression and gene duplication. For example, gene duplication and substrate binding site changes led to the evolution of the glucosinolate biosynthetic enzyme, AtIPMDH1, from a Leu biosynthetic enzyme. BAHD acyltransferases illustrate how enzymatic promiscuity leads to metabolite diversity. The examples 4-coumarate:CoA ligase and aromatic acid methyltransferases illustrate how promiscuity can potentiate the evolution of these specialized metabolic enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Effects of the DGAT1 polymorphism on test-day milk production traits throughout lactation

    DEFF Research Database (Denmark)

    Bovenhuis, Henk; Visker, H P W; van Valenberg, H J F

    2015-01-01

    Several studies have shown that the diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism has a major effect on milk production traits. It is less clear how effects of DGAT1 on milk production traits change throughout lactation, if dominance effects of DGAT1 are relevant, and whether DGAT1...... also affects lactose content, lactose yield, and total energy output in milk. Results from this study, using test-day records of 3 subsequent parities of around 1,800 cows, confirm previously reported effects of the DGAT1 polymorphism on milk, fat, and protein yield, as well as fat and protein content...

  11. Alteration of Lysophosphatidylcholine-Related Metabolic Parameters in the Plasma of Mice with Experimental Sepsis.

    Science.gov (United States)

    Ahn, Won-Gyun; Jung, Jun-Sub; Kwon, Hyeok Yil; Song, Dong-Keun

    2017-04-01

    Plasma concentration of lysophosphatidylcholine (LPC) was reported to decrease in patients with sepsis. However, the mechanisms of sepsis-induced decrease in plasma LPC levels are not currently well known. In mice subjected to cecal ligation and puncture (CLP), a model of polymicrobial peritoneal sepsis, we examined alterations in LPC-related metabolic parameters in plasma, i.e., the plasma concentration of LPC-related substances (i.e., phosphatidylcholine (PC) and lysophosphatidic acid (LPA)), and activities or levels in the plasma of some enzymes that can be involved in the regulation of plasma LPC concentration (i.e., secretory phospholipase A2 (sPLA2), lecithin:cholesterol acyltransferase (LCAT), acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), and autotaxin (ATX)), as well as plasma albumin concentration. We found that levels of LPC and albumin and enzyme activities of LCAT, ATX, and sPLA2 were decreased, whereas levels of PC, LPA, and LPCAT1-3 were increased in the plasma of mice subjected to CLP. Bacterial peritonitis led to alterations in all the measured LPC-related metabolic parameters in the plasma, which could potentially contribute to sepsis-induced decrease in plasma LPC levels. These findings could lead to the novel biomarkers of sepsis.

  12. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    Science.gov (United States)

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

  13. 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.

  14. Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome.

    Science.gov (United States)

    Wanders, R J; Barth, P G; van Roermund, C W; Ofman, R; Wolterman, R; Schutgens, R B; Tager, J M; van den Bosch, H; Bolhuis, P A

    1987-05-01

    In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.

  15. Serum lysophospholipid levels are altered in dyslipidemic hamsters.

    Science.gov (United States)

    Suárez-García, Susana; Caimari, Antoni; Del Bas, Josep Maria; Suárez, Manuel; Arola, Lluís

    2017-09-05

    Dyslipidemias are common disorders that predispose individuals to severe diseases. It is known that healthy living habits can prevent dyslipidemias if they are diagnosed properly. Therefore, biomarkers that assist in diagnosis are essential. The aim of this study was to identify biomarkers of dyslipidemia progression, which in turn disclose its etiology. These findings will pave the way for examinations of the regulatory mechanisms involved in dyslipidemias. Hamsters were fed either a normal-fat diet (NFD) or a high-fat diet. Some of the NFD-fed animals were further treated with the hyperlipidemic agent Poloxamer 407. Non-targeted metabolomics was used to investigate progressive changes in unknown serum metabolites. The hepatic expression of putative biomarker-related genes was also analyzed. The serum levels of lysophospholipids (Lyso-PLs) and their related enzymes lecithin-cholesterol acyltransferase (LCAT), secreted phospholipase A2 (sPLA2) and paraoxonase-1 were altered in dyslipidemic hamsters. Lysophosphatidylcholine levels were increased in diet-induced dyslipidemic groups, whereas lysophosphatidylethanolamine levels increased in response to the chemical treatment. The liver was significantly involved in regulating the levels of these molecules, based on the modified expression of endothelial lipase (Lipg), sPLA2 (Pla2g2a) and acyltransferases (Lcat and Lpcat3). We concluded that Lyso-PL evaluation could aid in the comprehensive diagnosis and management of lipid disorders.

  16. 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.

  17. Second-generation antisense oligonucleotides against β-catenin protect mice against diet-induced hepatic steatosis and hepatic and peripheral insulin resistance.

    Science.gov (United States)

    Popov, Violeta B; Jornayvaz, Francois R; Akgul, Emin O; Kanda, Shoichi; Jurczak, Michael J; Zhang, Dongyan; Abudukadier, Abulizi; Majumdar, Sachin K; Guigni, Blas; Petersen, Kitt Falk; Manchem, Vara Prasad; Bhanot, Sanjay; Shulman, Gerald I; Samuel, Varman T

    2016-03-01

    Although mutations in the Wnt/β-catenin signaling pathway are linked with the metabolic syndrome and type 2 diabetes in humans, the mechanism is unclear. High-fat-fed male C57BL/6 mice were treated for 4 wk with a 2'-O-methoxyethyl chimeric antisense oligonucleotide (ASO) to decrease hepatic and adipose expression of β-catenin. β-Catenin mRNA decreased by ≈80% in the liver and by 70% in white adipose tissue relative to control ASO-treated mice. β-Catenin ASO improved hepatic insulin sensitivity and increased insulin-stimulated whole body glucose metabolism, as assessed during hyperinsulinemic-euglycemic clamp in awake mice. β-Catenin ASO altered hepatic lipid composition in high-fat-fed mice. There were reductions in hepatic triglyceride (44%, P increase in ceramide content (P lipid content was attributed to decreased expression of sn-1,2 diacylglycerol acyltransferase and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase and an increase in serine palmitoyl transferase. The decrease in cellular diacyglycerol was associated with a 33% decrease in PKCε activation (P increase in Akt2 phosphorylation (P lipid-induced insulin resistance. © FASEB.

  18. Crystal Structure of Rat Carnitine Palmitoyltransferase II (CPT-II)

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao,Y.; Jogl, G.; Esser, V.; Tong, L.

    2006-01-01

    Carnitine palmitoyltransferase II (CPT-II) has a crucial role in the {beta}-oxidation of long-chain fatty acids in mitochondria. We report here the crystal structure of rat CPT-II at 1.9 Angstroms resolution. The overall structure shares strong similarity to those of short- and medium-chain carnitine acyltransferases, although detailed structural differences in the active site region have a significant impact on the substrate selectivity of CPT-II. Three aliphatic chains, possibly from a detergent that is used for the crystallization, were found in the structure. Two of them are located in the carnitine and CoA binding sites, respectively. The third aliphatic chain may mimic the long-chain acyl group in the substrate of CPT-II. The binding site for this aliphatic chain does not exist in the short- and medium-chain carnitine acyltransferases, due to conformational differences among the enzymes. A unique insert in CPT-II is positioned on the surface of the enzyme, with a highly hydrophobic surface. It is likely that this surface patch mediates the association of CPT-II with the inner membrane of the mitochondria.

  19. 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.

  20. The isolation and mapping of a novel hydroxycinnamoyltransferase in the globe artichoke chlorogenic acid pathway

    Science.gov (United States)

    Comino, Cinzia; Hehn, Alain; Moglia, Andrea; Menin, Barbara; Bourgaud, Frédéric; Lanteri, Sergio; Portis, Ezio

    2009-01-01

    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. PMID:19292932

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Arabidopsis ECERIFERUM2 Is a Component of the Fatty Acid Elongation Machinery Required for Fatty Acid Extension to Exceptional Lengths1[W][OA

    Science.gov (United States)

    Haslam, Tegan M.; Mañas-Fernández, Aurora; Zhao, Lifang; Kunst, Ljerka

    2012-01-01

    Primary aerial surfaces of land plants are coated by a lipidic cuticle, which forms a barrier against transpirational water loss and protects the plant from diverse stresses. Four enzymes of a fatty acid elongase complex are required for the synthesis of very-long-chain fatty acid (VLCFA) precursors of cuticular waxes. Fatty acid elongase substrate specificity is determined by a condensing enzyme that catalyzes the first reaction carried out by the complex. In Arabidopsis (Arabidopsis thaliana), characterized condensing enzymes involved in wax synthesis can only elongate VLCFAs up to 28 carbons (C28) in length, despite the predominance of C29 to C31 monomers in Arabidopsis stem wax. This suggests additional proteins are required for elongation beyond C28. The wax-deficient mutant eceriferum2 (cer2) lacks waxes longer than C28, implying that CER2, a putative BAHD acyltransferase, is required for C28 elongation. Here, we characterize the cer2 mutant and demonstrate that green fluorescent protein-tagged CER2 localizes to the endoplasmic reticulum, the site of VLCFA biosynthesis. We use site-directed mutagenesis to show that the classification of CER2 as a BAHD acyltransferase based on sequence homology does not fit with CER2 catalytic activity. Finally, we provide evidence for the function of CER2 in C28 elongation by an assay in yeast (Saccharomyces cerevisiae). PMID:22930748

  6. Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

    Science.gov (United States)

    Reynolds, Kyle B.; Taylor, Matthew C.; Zhou, Xue-Rong; Vanhercke, Thomas; Wood, Craig C.; Blanchard, Christopher L.; Singh, Surinder P.; Petrie, James R.

    2015-01-01

    Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation. PMID:25852716

  7. Modulation of Ingestive Behavior and Gastrointestinal Motility by Ghrelin in Diabetic Animals and Humans

    Directory of Open Access Journals (Sweden)

    Chih-Yen Chen

    2010-05-01

    Full Text Available Acyl ghrelin, a 28-amino acid peptide hormone, is the endogenous cognate ligand for the growth hormone secretagogue receptor. Ghrelin is involved in stimulating growth hormone release, eliciting feeding behavior, inducing adiposity and stimulating gastrointestinal motility. Ghrelin is unique for its post-translational modification of O-n-octanoylation at serine 3 through ghrelin O-acyltransferase, and is the only peripheral signal to enhance food intake. Plasma ghrelin levels manifest “biphasic changes” in diabetes mellitus (DM. In the early stage of DM, the stomach significantly increases the secretion of ghrelin into the plasma, and elevated plasma ghrelin levels are correlated with diabetic hyperphagic feeding and accelerated gastrointestinal motility. In the late stage of DM, plasma ghrelin levels may be lower, which might be linked with anorexia/muscle wasting, delayed gastrointestinal transit, and even gastroparesis. Therefore, the unique ghrelin system may be the most important player compared to the other hindgut hormones participating in the “entero-insular axis”. Further studies using either knockdown or knockout of ghrelin gene products and ghrelin O-acyltransferase may unravel the pathogenesis of DM, and show benefits in combating this disease and metabolic syndrome.

  8. Metabolic Effects of Clenbuterol and Salbutamol on Pork Meat Studied Using Internal Extractive Electrospray Ionization Mass Spectrometry.

    Science.gov (United States)

    Lu, Haiyan; Zhang, Hua; Zhu, Tenggao; Xiao, Yipo; Xie, Shaoxian; Gu, Haiwei; Cui, Meng; Luo, Liping

    2017-07-11

    Direct mass spectrometry analysis of metabolic effects of clenbuterol and salbutamol on pork quality at the molecular level is incredibly beneficial for food regulations, public health and the development of new anti-obesity drugs. With internal extractive electrospray ionization mass spectrometry (iEESI-MS), nutrients including creatine, amino acids, L-carnitine, vitamin B6, carnosine and phosphatidylcholines in pork tissue were identified, without sample pretreatment, using collision-induced dissociation (CID) experiments and by comparison with authentic compounds. Furthermore, normal pork samples were clearly differentiated from pork samples with clenbuterol and salbutamol via principal component analysis (PCA). Correlation analysis performed on the spectral data revealed that the above-mentioned nutrients strongly correlated with pork quality, and the absolute intensity of phosphatidylcholines in normal pork was much higher than pork contaminated by clenbuterol and salbutamol. Our findings suggested that clenbuterol and salbutamol may render effects on the activity of carnitine acyltransferase I, hence the process that L-carnitine transports long-chain fatty acids into mitochondria and the formation of phosphatidylcholines might be affected. However, the underlying metabolic mechanisms of clenbuterol and salbutamol on carnitine acyltransferase I requires more comprehensive studies in future work.

  9. 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.

  10. Absence of TRH receptor 1 in male mice affects gastric ghrelin production.

    Science.gov (United States)

    Mayerl, Steffen; Liebsch, Claudia; Visser, Theo J; Heuer, Heike

    2015-02-01

    TRH not only functions as a thyrotropin releasing hormone but also acts as a neuropeptide in central circuits regulating food intake and energy expenditure. As one suggested mode of action, TRH expressed in the caudal brainstem influences vagal activity by activating TRH receptor 1 (TRH-R1). In order to evaluate the impact of a diminished medullary TRH signaling on ghrelin metabolism, we analyzed metabolic changes of TRH-R1 knockout (R1ko) mice in response to 24 hours of food deprivation. Because R1ko mice are hypothyroid, we also studied eu- and hypothyroid wild-type (wt) animals and R1ko mice rendered euthyroid by thyroid hormone treatment. Independent of their thyroidal state, R1ko mice displayed a higher body weight loss than wt animals and a delayed reduction in locomotor activity upon fasting. Ghrelin transcript levels in the stomach as well as total ghrelin levels in the circulation were equally high in fasted wt and R1ko mice. In contrast, only wt mice responded to fasting with a rise in ghrelin-O-acyltransferase mRNA expression and consequently an increase in serum levels of acylated ghrelin. Together, our data suggest that an up-regulation of medullary TRH expression and subsequently enhanced activation of TRH-R1 in the vagal system represents a critical step in the stimulation of ghrelin-O-acyltransferase expression upon starvation that in turn is important for adjusting the circulating levels of acylated ghrelin to the fasting condition.

  11. 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.

    2014-01-01

    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. PMID:25058047

  12. Design, synthesis and biological evaluation of sugar-derived esters, [alpha]-ketoesters and [alpha]-ketoamides as inhibitors for Mycobacterium tuberculosis antigen 85C

    Energy Technology Data Exchange (ETDEWEB)

    Sanki, Aditya K.; Boucau, Julie; Umesiri, Francis E.; Ronning, Donald R.; Sucheck, Steven J.

    2010-08-16

    Peptide-based 1,2-dicarbonyl compounds have emerged as potent inhibitors for serine proteases. Herein, we have designed and synthesized D-arabinose and D-trehalose-based esters, {alpha}-ketoesters and {alpha}-ketoamides, and evaluated their inhibitory activity against Mycobacterium tuberculosis (Mtb) antigen 85C (ag85C), an acyltransferase in the serine hydrolase superfamily. In addition the compounds were evaluated for the ability to inhibit the growth of Mycobacterium smegmatis ATCC 14468, a non-pathogenic surrogate for Mtb. Among the synthetic analogs evaluated only the methyl ester1 derived from D-arabinose was found to inhibit the acyltransferase activity of ag85C (IC{sub 50} = 25 mM). Based on this weak inhibitory activity it was not surprising that none of the compounds inhibits the growth of M. smegmatis. In spite of the weak inhibitory activity of 1, X-ray crystallography on crystals of ag85C soaked with 1 suggested the formation of a covalent ester adduct between 1 and the Ser124 side chain hydroxyl moiety found within the catalytic site of ag85C; however, some of the active site electron density appears to result from bound glycerol. The lack of activity associated with the {alpha}-ketoester and {alpha}-ketoamide derivatives of D-trehalose may be the result of intramolecular cyclization of the {alpha}-keto moiety with the nearby C-4/4' hydroxyls leading to the formation of stable bicyclo-ester and amide derivatives.

  13. RNAi targeting putative genes in phosphatidylcholine turnover results in significant change in fatty acid composition in Crambe abyssinica seed oil.

    Science.gov (United States)

    Guan, Rui; Li, Xueyuan; Hofvander, Per; Zhou, Xue-Rong; Wang, Danni; Stymne, Sten; Zhu, Li-Hua

    2015-04-01

    The aim of this study was to evaluate the importance of three enzymes, LPCAT, PDCT and PDAT, involved in acyl turnover in phosphatidylcholine in order to explore the possibility of further increasing erucic acid (22:1) content in Crambe seed oil. The complete coding sequences of LPCAT1-1 and LPCAT1-2 encoding lysophosphatidylcholine acyltransferase (LPCAT), PDCT1 and PDCT2 encoding phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), and PDAT encoding phospholipid:diacylglycerol acyltransferase (PDAT) were cloned from developing Crambe seeds. The alignment of deduced amino acid sequences displayed a high similarity to the Arabidopsis homologs. Transgenic lines expressing RNA interference (RNAi) targeting either single or double genes showed significant changes in the fatty acid composition of seed oil. An increase in oleic acid (18:1) was observed, to varying degrees, in all of the transgenic lines, and a cumulative effect of increased 18:1 was shown in the LPCAT-PDCT double-gene RNAi. However, LPCAT single-gene RNAi led to a decrease in 22:1 accumulation, while PDCT or PDAT single-gene RNAi had no obvious effect on the level of 22:1. In agreement with the abovementioned oil phenotypes, the transcript levels of the target genes in these transgenic lines were generally reduced compared to wild-type levels. In this paper, we discuss the potential to further increase the 22:1 content in Crambe seed oil through downregulation of these genes in combination with fatty acid elongase and desaturases.

  14. In vivo incorporation of lauric acid into rat adipose tissue triacylglycerols.

    Science.gov (United States)

    Bugaut, M

    1989-03-01

    An in vivo approach was taken to examine fatty acid esterification in adipose tissue using a coconut oil-enriched diet. Rats were fed a diet containing coconut oil (50% lauric acid) for six weeks. Triacylglycerols from perirenal adipose tissue were fractionated by silver nitrate-thin layer chromatography and, then, preparative gas chromatography. The distribution of 169 triacylglycerol types accounting for 97% of total triacylglycerols was determined. There was evidence for a very high content of mixed triacylglycerols composed of intermediate (12:0 and 14:0) and long acyl moieties. No significant differences were observed between the experimental distribution of triacylglycerol types and the random distribution, calculated from the total fatty acid composition. This indicated that most long chain triacylglycerols stored before coconut oil feeding would have been rearranged after the six weeks of coconut oil feeding. The experimental proportion of trilauroylglycerol reached 2%, as expected from its random proportion, and the proportions of dilauroylacylglycerols were slightly higher than the random values. Present results were compared with those previously obtained from triacylglycerols of adipose tissue of rats fed a low-fat standard diet. From our results and those of other authors, it is suggested that lauric acid is a good substrate for sn-glycero-3-phosphate acyltransferase and diacylglycerol acyltransferase in rat adipose tissue.

  15. Cloning and characterization of GPAT gene from Lepidium latifolium L.: a step towards translational research in agri-genomics for food and fuel.

    Science.gov (United States)

    Gupta, Sanjay Mohan; Pandey, Pankaj; Grover, Atul; Patade, Vikas Yadav; Singh, Sadhana; Ahmed, Zakwan

    2013-07-01

    Glycerol-3-phosphate acyltransferase (GPAT) catalyzes first and the rate limiting step in glycerolipid synthesis pathway, which in turn contribute to stabilization of plasma membrane structure and oil lipid synthesis in plant cells. Here, we report cloning and characterization of GPAT gene from Lepidium latifolium (LlaGPAT). The cDNA sequence (1,615 bp) of LlaGPAT gene consisted of 1,113 bp ORF encoding a protein of 370 aa residues, with deduced mass of 41.2 kDa and four acyltransferase (AT) motifs having role in catalysis and in glycerol-3-phosphate binding. Southern blot analysis suggested presence of a single copy of the gene in the genome. Tissue specific expression of the gene was seen more abundantly in aerial parts, compared to the roots. Quantitative real-time PCR indicated down-regulation of the gene by cold (4 °C), drought (PEG6000), salt (300 mM NaCl) and ABA (100 μM) treatments. Considering the vitality of the function of encoded enzyme, LlaGPAT can be considered a potential candidate gene for genetic engineering of oil yields and abiotic stress management in food as well as fuel crops.

  16. Effects of cigarette smoking on HDL quantity and function: implications for atherosclerosis.

    Science.gov (United States)

    He, Bai-mei; Zhao, Shui-ping; Peng, Zhen-yu

    2013-11-01

    Cigarette smoking has been identified as an independent and preventable risk factor for atherosclerosis and cardiovascular disease. Population studies have shown that plasma high density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing cardiovascular disease. Cigarette smoking is associated with reduced HDL cholesterol levels. Cigarette smoking can alter the critical enzymes of lipid transport, lowering lecithin: cholesterol acyltransferase (LCAT) activity and altering cholesterol ester transfer protein (CETP) and hepatic lipase activity, which attributes to its impact on HDL metabolism and HDL subfractions distribution. In addition, HDL is susceptible to oxidative modifications by cigarette smoking, which makes HDL become dysfunctional and lose its atheroprotective properties in smokers. Therefore, cigarette smoking has a negative impact on both HDL quantity and function, which can explain, in part, the increased risk of cardiovascular disease in smokers. © 2013 Wiley Periodicals, Inc.

  17. Using self-organizing map (SOM) and support vector machine (SVM) for classification of selectivity of ACAT inhibitors.

    Science.gov (United States)

    Wang, Ling; Wang, Maolin; Yan, Aixia; Dai, Bin

    2013-02-01

    Using a self-organizing map (SOM) and support vector machine, two classification models were built to predict whether a compound is a selective inhibitor toward the two Acyl-coenzyme A: cholesterol acyltransferase (ACAT) isozymes, ACAT-1 and ACAT-2. A dataset of 97 ACAT inhibitors was collected. For each molecule, the global descriptors, 2D and 3D property autocorrelation descriptors and autocorrelation of surface properties were calculated from the program ADRIANA.Code. The prediction accuracies of the models (based on the training/ test set splitting by SOM method) for the test sets are 88.9 % for SOM1, 92.6 % for SVM1 model. In addition, the extended connectivity fingerprints (ECFP_4) for all the molecules were calculated and the structure-activity relationship of selective ACAT inhibitors was summarized, which may help find important structural features of inhibitors relating to the selectivity of ACAT isozymes.

  18. Polyacetylenic compounds, ACAT inhibitors from the roots of Panax ginseng.

    Science.gov (United States)

    Rho, Mun-Chual; Lee, Hyun Sun; Lee, Seung Woong; Chang, Jong Sun; Kwon, Oh Eok; Chung, Mi Yeon; Kim, Young Kook

    2005-02-23

    Acyl-CoA: cholesterol acyltransferase (ACAT), which plays a role in the absorption, storage, and production of cholesterol, has been explored as a potential target for pharmacological intervention of hyperlipidemia and atherosclerotic disease. In our search for ACAT inhibitors from natural sources, the petroleum ether extract of Panax ginseng showed moderate inhibition of ACAT enzyme from rat liver microsomes. Bioactivity-guided fractionations led to the isolation of one new polyacetylenic compound, (9R,10S)-epoxy-16-heptadecene-4, 6-diyne-3-one (1), in addition to the previously reported polyacetylenic compounds 2 and 3. Their chemical structures were elucidated on the basis of spectroscopic evidence (UV, IR, NMR, and MS). The compounds 1, 2, and 3 showed significant ACAT inhibition with IC(50) values of 35, 47, and 21 microM, respectively.

  19. ACAT inhibition and amyloid beta reduction.

    Science.gov (United States)

    Bhattacharyya, Raja; Kovacs, Dora M

    2010-08-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Abeta) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Abeta production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Abeta generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Abeta generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD. Copyright 2010 Elsevier B.V. All rights reserved.

  20. Abrogating cholesterol esterification suppresses growth and metastasis of pancreatic cancer

    Science.gov (United States)

    Li, J; Gu, D; Lee, S S-Y; Song, B; Bandyopadhyay, S; Chen, S; Konieczny, S F; Ratliff, T L; Liu, X; Xie, J; Cheng, J-X

    2016-01-01

    Cancer cells are known to execute reprogramed metabolism of glucose, amino acids and lipids. Here, we report a significant role of cholesterol metabolism in cancer metastasis. By using label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester in human pancreatic cancer specimens and cell lines, mediated by acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme. Expression of ACAT-1 showed a correlation with poor patient survival. Abrogation of cholesterol esterification, either by an ACAT-1 inhibitor or by shRNA knockdown, significantly suppressed tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Mechanically, ACAT-1 inhibition increased intracellular free cholesterol level, which was associated with elevated endoplasmic reticulum stress and caused apoptosis. Collectively, our results demonstrate a new strategy for treating metastatic pancreatic cancer by inhibiting cholesterol esterification. PMID:27132508

  1. 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.

  2. [Effect of five kinds of vegetable seed oil on serum lipid and lipid peroxidation in rats].

    Science.gov (United States)

    Guo, Y; Cai, X; Zhao, X; Shi, R

    2001-01-01

    The effects of vegetable seed oil on hyperlipidemia induced by high lipid diet in rats. Male adult Wistar rats were fed on the test diet containing 94% high lipid diet and 6% lard pinon seed oil, perilla seed oil, blackcurrent seed oil, borage seed oil and evening primrose seed oil respectively for 3 weeks. The results showed that the vale of trilyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol (LDL-C), LDL-C/HDL-C(high density lipoprotein cholesterol) ratio increased and the vale of HDL-C/TC ratio and lecithin-cholesterol acyltransferase(LCAT) activity decreased in the groups with vegetable seed oil were less than that of the control group. The results suggested that all the five kinds of vegetable seed oil had the effect of regulating lipid metabolism of hyperlipidemia rats to some extent. Pinon seed oil and borage seed oil may be well suited for the prevention of atherosclerosis.

  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. [Chromatographic fractionation of cholesterol esters in Morgagni's hepatic cirrhosis. Preliminary note].

    Science.gov (United States)

    Ceccanti, M; Romeo, M; Carlomusto, C; Santini, P; Tagliamonte, L

    1978-11-14

    The behaviour of plasma cholesteryl esters has been investigated in patients with liver cirrhosis (so-called Laennec's cirrhosis). Both absolute and percentage values of esters containing di-, tri- and tetra-unsaturated fatty acids were decreased below the normal range; however, the various esters groups showed unequal decrement rates. These findings may be the result of a low lecithin: cholesterol-acyltransferase (LCAT) activity, due either to depression of the enzyme syntesis in the liver, or to inadequate substrate supply (possibly related with an impaired fatty acid production and lecithin synthesis). A decrease of all ester fractions, or a selective one, could also be induced by the releasing of abnormal hydrolases from damaged liver tissues.

  5. Biochemistry of Apple Aroma: A Review

    Directory of Open Access Journals (Sweden)

    Miguel Espino-Díaz

    2016-01-01

    Full Text Available Flavour is a key quality att ribute 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.

  6. Lipid droplets and lipotoxicity during autophagy.

    Science.gov (United States)

    Nguyen, Truc B; Olzmann, James A

    2017-08-14

    Lipid droplets (LDs) are neutral lipid storage organelles that provide a rapidly accessible source of fatty acids (FAs) for energy during periods of nutrient deprivation. Surprisingly, lipids released by the macroautophagic/autophagic breakdown of membranous organelles are packaged and stored in new LDs during periods of prolonged starvation. Why cells would store FAs during an energy crisis was unknown. In our recent study, we demonstrated that FAs released during MTORC1-regulated autophagy are selectively channeled by DGAT1 (diacylglycerol O-acyltransferase 1) into triacylglycerol (TAG)-rich LDs. These DGAT1-dependent LDs sequester FAs and prevent the accumulation of acylcarnitines, which otherwise directly disrupt mitochondrial integrity. Our findings establish LD biogenesis as a general cellular response to periods of high autophagic flux that provide a lipid buffering system to mitigate lipotoxic cellular damage.

  7. A Review of Selected Genes with Known Effects on Performance and Health of Cattle

    Directory of Open Access Journals (Sweden)

    Eduardo Casas

    2016-12-01

    Full Text Available 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 (BLAD, Deficiency of the Uridine Monophosphate Synthase (DUMPS, Bovine Chronic Interstitial Nephritis (BCIN, 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, Diaglycerol Acyltransferase, Syndactyly, Maple Syrup Urine Disease, Slick Hair, Factor XI Deficiency, and Mu-Calpain. This review is not meant to be comprehensive, and relevant information is provided to ascertain genetic markers associated with the conditions.

  8. Downregulation of Hepatic De Novo Lipogenesis and Adipogenesis in Adipocytes by Pinus densiflora Bark Extract.

    Science.gov (United States)

    Ahn, Hyemyoung; Jeong, Jeongho; Moyo, Knowledge; Ryu, Yungsun; Min, Bokkee; Yun, Seong Ho; Kim, Hwa Yeon; Kim, Wooki; Go, Gwang-Woong

    2017-11-28

    Korean red pine (Pinus densiflora) bark extract, PineXol (PX), was investigated for its potential antioxidant and anti-inflammation effects in vitro. It was hypothesized that PX treatment (25-150 μg/ml) would reduce the lipid synthesis in HepG2 hepatocytes as well as lipid accumulation in 3T3-L1 adipocytes. Hepatocytes' intracellular triglycerides and cholesterol were decreased in the PX 150 μg/ml treatment group compared with the control (p novo lipogenic proteins (acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, elongase of very long chain fatty acids 6, glycerol-3-phosphate acyltransferase 1, and sterol regulatory element-binding protein 1) were significantly decreased in hepatocytes by PX 150 μg/ml treatment compared with the control (p lipogenesis and by inhibiting adipogenesis in adipocytes.

  9. Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus.

    Science.gov (United States)

    Facciotti, Federica; Ramanjaneyulu, Gundimeda S; Lepore, Marco; Sansano, Sebastiano; Cavallari, Marco; Kistowska, Magdalena; Forss-Petter, Sonja; Ni, Guanghui; Colone, Alessia; Singhal, Amit; Berger, Johannes; Xia, Chengfeng; Mori, Lucia; De Libero, Gennaro

    2012-03-18

    The development and maturation of semi-invariant natural killer T cells (iNKT cells) rely on the recognition of self antigens presented by CD1d restriction molecules in thymus. The nature of the stimulatory thymic self lipids remains elusive. We isolated lipids from thymocytes and found that ether-bonded mono-alkyl glycerophosphates and the precursors and degradation products of plasmalogens stimulated iNKT cells. Synthetic analogs showed high potency in activating thymic and peripheral iNKT cells. Mice deficient in the peroxisomal enzyme glyceronephosphate O-acyltransferase (GNPAT), essential for the synthesis of ether lipids, had significant alteration of the thymic maturation of iNKT cells and fewer iNKT cells in both thymus and peripheral organs, which confirmed the role of ether-bonded lipids as iNKT cell antigens. Thus, peroxisome-derived lipids are nonredundant self antigens required for the generation of a full iNKT cell repertoire.

  10. 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.

  11. Effect of tofacitinib on lipid levels and lipid-related parameters in patients with moderate to severe psoriasis

    DEFF Research Database (Denmark)

    Wolk, Robert; Armstrong, Ehrin J; Hansen, Peter R

    2017-01-01

    BACKGROUND: Psoriasis is a systemic inflammatory disease associated with increased cardiovascular (CV) risk and altered lipid metabolism. Tofacitinib is an oral Janus kinase inhibitor. OBJECTIVE: The aim of the study was to investigate the effects of tofacitinib on traditional and nontraditional...... cholesterol levels, triglycerides, lipoproteins, lipid particles, lipid-related parameters/CV risk markers, and high-density lipoprotein (HDL) function analyses. RESULTS: At week 16, small concurrent increases in mean low-density lipoprotein cholesterol (LDL-C) and HDL cholesterol (HDL-C) levels were observed......, and HDL-associated serum amyloid A, which reduces the anti-atherogenic potential of HDL, decreased. HDL capacity to promote cholesterol efflux from macrophages did not change. Lecithin-cholesterol acyltransferase activity, which is associated with reverse cholesterol transport, increased. Markers...

  12. AcEST: BP915294 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Mus musculus Align length 42 Score (bit) 33.9 E-value 0.29 Report BLASTX 2.2.19 [Nov-02-2008] Reference: Alt...s Searching..................................................done Score E Sequences producing significant al...-3-phosphate acyltransferase-like protein 1 OS=Mus musculus PE=2 SV=2 Length = 801 Score = 33.9 bits (76), E... Twist) GN=ileS PE=3 SV=2 Length = 1056 Score = 30.4 bits (67), Expect = 3.2 Identities = 14/39 (35%), Posit...OW8 Isoleucyl-tRNA synthetase OS=Tropheryma whipplei (strain TW08/27) GN=ileS PE=3 SV=1 Length = 1056 Score

  13. Augmentation of sebaceous lipogenesis by an ethanol extract of Grifola frondosa (Maitake mushroom) in hamsters in vivo and in vitro.

    Science.gov (United States)

    Nagao, Mie; Sato, Takashi; Akimoto, Noriko; Kato, Yuya; Takahashi, Masao; Ito, Akira

    2009-08-01

    Grifola frondosa (Maitake mushroom) is an edible and medicinal mushroom with versatile effects such as antitumor and immunomodulating actions. Here, we demonstrated that an ethanol extract of G. frondosa fruiting body (Maitake extract) augmented intracellular lipid droplet formation and the production of triacylglycerols (TG), a major component of sebum, along with the activation of diacylglycerol acyltransferase, a rate-limiting enzyme of TG synthesis in cultured hamster sebocytes. The topical treatment of Maitake extract on the skin of hamster auricles augmented sebum accumulation in sebaceous glands and ducts. However, in comparison with the Maitake extract, another ethanol extract prepared from Agaricus blazei Murill showed less activity in sebaceous lipogenesis in hamsters in vivo and in vitro. These results provide novel evidence that Maitake extract augments sebaceous lipogenesis in hamsters in vivo and in vitro. Thus, Maitake extract is likely to be a unique agent leading to the remission of dry skin.

  14. Cloning and heterologous expression of the penicillin biosynthetic gene cluster from penicillum chrysogenum.

    Science.gov (United States)

    Smith, D J; Burnham, M K; Edwards, J; Earl, A J; Turner, G

    1990-01-01

    A cosmid clone containing the putative penicillin biosynthetic gene cluster from Penicillium chrysogenum was used to transform the related filamentous fungi Neurospora crassa and Aspergillus niger, which do not produce beta-lactam antibiotics. Both of the transformed hosts contained intact P. chrysogenum DNA derived from the cosmid clone and produced authentic penicillin V. Assays of penicillin biosynthetic enzyme activity additionally demonstrated that they possessed delta-(L-alpha-amino-adipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthetase (IPNS) and acyl coenzyme A:6-aminopenicillanic acid acyltransferase (ACT) activity. The data suggests that genes encoding all the enzymes necessary for the biosynthesis of penicillin from amino acid precursors are closely linked in P. chrysogenum and constitute a gene cluster.

  15. 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...

  16. 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....

  17. Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.

    Science.gov (United States)

    Fan, Pengxiang; Miller, Abigail M; Liu, Xiaoxiao; Jones, A Daniel; Last, Robert L

    2017-12-12

    Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

  18. Testosterone conjugating activities in invertebrates: are they targets for endocrine disruptors?

    Science.gov (United States)

    Janer, G; Sternberg, R M; LeBlanc, G A; Porte, C

    2005-02-10

    Testosterone conjugation activities, microsomal acyltransferases and cytosolic sulfotransferases, were investigated in three invertebrate species, the gastropod Marisa cornuarietis, the amphipod Hyalella azteca, and the echinoderm Paracentrotus lividus. The goals of the study were to characterize steroid conjugation pathways in different invertebrate phyla and to assess the susceptibility of those processes to disruption by environmental chemicals. All three species exhibited palmitoyl-CoA: testosterone acyltransferase activity (ATAT) in the range of 100-510 pmol/min/mg protein. Despite similarities in specific activities, kinetic studies indicated that ATAT had a higher affinity for testosterone but a lower V(max) in M. cornuarietis than in P. lividus, and intermediate values were found for H. azteca. In contrast, the activity of testosterone sulfotransferase (SULT) was rather low (0.05-0.18 pmol/min/mg protein) in M. cornuarietis and H. azteca. The low activity precluded kinetic analyses and inhibition studies with these species. P. lividus digestive tube displayed high SULT activity (50-170 pmol/min/mg protein) at moderate testosterone concentrations, but was inhibited at high testosterone concentrations. The interference of model pollutants (triphenyltin (TPT), tributyltin (TBT), and fenarimol) with these conjugation pathways was investigated in vitro. Both TPT and TBT (100 microM) inhibited ATAT in P. lividus (68 and 42% inhibition, respectively), and appeared to act as non-competitive inhibitors. ATAT activity in M. cornuarietis was less affected by organotins, and a significant inhibition (20% inhibition) was detected only with TBT. Fenarimol (100 microM) did not affect ATAT in any of the species tested. Sulfation of testosterone was suppressed by the organotins as well as fenarimol when using cytosolic preparations from P. lividus. These results demonstrated the existence of interphyla differences in testosterone conjugation, and revealed that these

  19. Phosphatidylcholine formation by LPCAT1 is regulated by Ca2+ and the redox status of the cell

    Directory of Open Access Journals (Sweden)

    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.

  20. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    Science.gov (United States)

    Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

    2014-01-01

    Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

  1. HMG-CoA reductase, cholesterol 7alpha-hydroxylase, LCAT, ACAT, LDL receptor, and SRB-1 in hereditary analbuminemia.

    Science.gov (United States)

    Liang, Kaihui; Vaziri, Nosratola D

    2003-07-01

    Hereditary analbuminemia is associated with hypercholesterolemia, which has been shown to be primarily caused by increased extrahepatic production of cholesterol. Nagase rats with hereditary analbuminemia (NAR) have been used as a model to dissect the effect of primary hypoalbuminemia from that caused by proteinuria in nephrotic syndrome. The present study was undertaken to explore the effect of hereditary analbuminemia on protein expression of the key factors involved in cholesterol metabolism. Hepatic tissue protein abundance of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7alpha-hydroxylase (a rate-limiting enzyme in cholesterol catabolism), low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor (SRB-1), acyl-coA cholesterol acyltransferase-2 (ACAT-2), and plasma concentration of lecithin cholesterol acyltransferase (LCAT), as well as HMG-CoA reductase, ACAT, and LCAT activities were determined in fasting male NAR and Sprague-Dawley control rats. The NAR group exhibited significant up-regulation of HMG-CoA reductase protein abundance but normal HMG-CoA reductase enzymatic activity. This was coupled with a significant up-regulation of cholesterol 7alpha-hydroxylase and a mild up-regulation of ACAT protein abundance and activity. However, hepatic LDL receptor and HDL receptor and plasma LCAT protein concentration and activity were normal in NAR. Hypercholesterolemia in NAR is associated with elevated hepatic HMG-CoA reductase protein abundance, but normal HMG-CoA reductase activity. These findings point to post-translational regulation of this enzyme and favor an extrahepatic origin of hypercholesterolemia in NAR. The observed up-regulation of cholesterol 7alpha-hydroxylase represents a compensatory response to the associated hypercholesterolemia. Unlike nephrotic syndrome, which causes severe LDL receptor, HDL receptor, and LCAT deficiencies, hereditary analbuminemia does not affect these proteins.

  2. Lipidomic and spatio-temporal imaging of fat by mass spectrometry in mice duodenum during lipid digestion.

    Directory of Open Access Journals (Sweden)

    Alexandre Seyer

    Full Text Available 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.

  3. Bovine gene polymorphisms related to fat deposition and meat tenderness

    Directory of Open Access Journals (Sweden)

    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.

  4. Tissue- and sex-specific effects of β-carotene 15,15' oxygenase (BCO1) on retinoid and lipid metabolism in adult and developing mice.

    Science.gov (United States)

    Kim, Youn-Kyung; Zuccaro, Michael V; Costabile, Brianna K; Rodas, Rebeka; Quadro, Loredana

    2015-04-15

    In mammals, β-carotene-15,15'-oxygenase (BCO1) is the main enzyme that cleaves β-carotene, the most abundant vitamin A precursor, to generate retinoids (vitamin A derivatives), both in adult and developing tissues. We previously reported that, in addition to this function, BCO1 can also influence the synthesis of retinyl esters, the storage form of retinoids, in the mouse embryo at mid-gestation. Indeed, lack of embryonic BCO1 impaired both lecithin-dependent and acyl CoA-dependent retinol esterification, mediated by lecithin:retinol acyltransferase (LRAT) and acyl CoA:retinol acyltransferase (ARAT), respectively. Furthermore, embryonic BCO1 also influenced the ester pools of cholesterol and diacylglycerol. In this report, we gained novel insights into this alternative function of BCO1 by investigating whether BCO1 influenced embryonic retinoid and lipid metabolism in a tissue-dependent manner. To this end, livers and brains from wild-type and BCO1-/- embryos at mid-gestation were analyzed for retinoid and lipid content, as well as gene expression levels. We also asked whether or not the role of BCO1 as a regulator of lecithin- and acyl CoA-dependent retinol esterification was exclusively restricted to the developing tissues. Thus, a survey of retinol and retinyl ester levels in adult tissues of wild-type, BCO1-/-, LRAT-/- and LRAT-/-BCO1-/- mice was performed. We showed that the absence of BCO1 affects embryonic retinoid and lipid homeostasis in a tissue-specific manner and that retinyl ester formation is also influenced by BCO1 in a few adult tissues (pancreas, lung, heart and adipose) in a sex-dependent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Esterification and hydrolysis of vitamin A in the liver of brook trout (Salvelinus fontinalis) and the influence of a coplanar polychlorinated biphenyl

    Energy Technology Data Exchange (ETDEWEB)

    Ndayibagira, A.; Spear, P.A. [Centre de Recherche TOXEN and Departement des Sciences Biologiques, Universite du Quebec a Montreal, C.P. 8888, succursale Centre Ville Montreal (Canada)

    1999-03-01

    Recent reports of extremely low retinoid stores in fish living in contaminated river systems prompted an initial investigation of the mechanisms of hepatic storage and mobilization in brook trout. Enzyme characterization in microsomes revealed a lecithin:retinol acyltransferase activity (LRAT) optimum in the alkaline range (pH 9.0; V{sub max}=0.6 nmol per mg prot. h{sup -1}; K{sub m}=10.2 {mu}M) which is not known to occur in mammals, in addition to a secondary optimum at pH 6.5 typical of mammals. Acyl CoA:retinol acyltransferase (ARAT) kinetic parameters were quite different to those of mammals. The substrate affinity of trout ARAT (K{sub m}=1.6 {mu}M) was approximately 22-fold greater than that of the rat while maximal velocity (V{sub max}=0.2 nmol per mg prot. h{sup -1}) was 18-fold less. Retinyl ester hydrolase activity (REH) was optimal under acid conditions (pH 4.2; V{sub max}=6.6 nmol per mg prot. h{sup -1}; K{sub m}=0.6 mM), was inhibited by a bile salt analogue and was greater in males than females. This REH was tentatively categorized as a bile salt-independent, acid retinyl ester hydrolase (BSI-AREH). REH was inhibited in a dose-dependent manner following in vivo exposure to a representative environmental contaminant the coplanar polychlorinated biphenyl (PCB), 3,3minutes or feet,4,4minutes or feet-tetrachlorobiphenyl (TCBP). Inhibition may be an indirect effect because enzyme activity was not affected by in vitro exposure of control microsomes. REH inhibition in the brook trout may affect the uptake of retinyl esters (REs) from chylomicron remnants as well as the mobilization of stored REs. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  6. Responsibility of regulatory gene expression and repressed protein synthesis for triacylglycerol accumulation on sulfur-starvation in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    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.

  7. Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2α-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli

    Science.gov (United States)

    Walker, Kevin; Croteau, Rodney

    2000-01-01

    A cDNA clone encoding a taxane 2α-O-benzoyltransferase has been isolated from Taxus cuspidata. The recombinant enzyme catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus appears to function in a late-stage acylation step of the Taxol biosynthetic pathway. By employing a homology-based PCR cloning strategy for generating acyltransferase oligodeoxynucleotide probes, several gene fragments were amplified and used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Escherichia coli. The functionally expressed benzoyltransferase was confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verification of the product, 7,13-diacetylbaccatin III, derived from 2-debenzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates in the corresponding cell-free extract. The full-length cDNA has an open reading frame of 1,320 base pairs and encodes a protein of 440 residues with a molecular weight of 50,089. The recombinant benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respectively, and is apparently regiospecific for acylation of the 2α-hydroxyl group of the functionalized taxane nucleus. This enzyme may be used to improve the production yields of Taxol and for the semisynthesis of drug analogs bearing modified aroyl groups at the C2 position. PMID:11095755

  8. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. 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. PMID:26745266

  10. Drosophila Courtship Conditioning As a Measure of Learning and Memory.

    Science.gov (United States)

    Koemans, Tom S; Oppitz, Cornelia; Donders, Rogier A T; van Bokhoven, Hans; Schenck, Annette; Keleman, Krystyna; Kramer, Jamie M

    2017-06-05

    Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular

  11. DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules

    Directory of Open Access Journals (Sweden)

    Maria Kwiatkowska

    2012-10-01

    Full Text Available The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2 antibody revealed in
    A. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as well
    as in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presence
    of microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around the
    lipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.
    Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participated
    in lipid synthesis. A similar phenomenon may take place in A. thaliana.The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2 antibody revealed in
    A. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as well
    as in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presence
    of microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around the
    lipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations.
    Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participated
    in lipid synthesis. A similar phenomenon may take place in A. thaliana.

  12. DNA adducts from nitroreduction of 2,7-dinitrofluorene, a mammary gland carcinogen, catalyzed by rat liver or mammary gland cytosol.

    Science.gov (United States)

    Ritter, Clare L; Culp, Sandra J; Freeman, James P; Marques, M Matilde; Beland, Frederick A; Malejka-Giganti, Danuta

    2002-04-01

    Nitrofluorenes are mutagenic and carcinogenic environmental pollutants arising chiefly from combustion of fossil fuels. Nitro aromatic compounds undergo nitroreduction to N-hydroxy arylamines that bind to DNA directly or after O-esterification. This study analyzes the DNA binding and adducts from the in vitro nitroreduction of 2,7-dinitrofluorene (2,7-diNF), a potent mammary carcinogen in the rat. Potential adduct(s) of 2,7-diNF was (were) generated by reduction of 2-nitroso-7-NF with ascorbate/H(+) in the presence of calf thymus DNA. The major adduct was characterized by HPLC/ESI/MS and (1)H NMR spectrometry as N-(deoxyguanosin-8-yl)-2-amino-7-NF, and a minor one was determined by HPLC/ESI/MS to be a deoxyadenosine adduct of 2-amino-7-NF. Products from enzymatic nitroreduction were monitored by HPLC and DNA adduct formation by (32)P-postlabeling. Xanthine oxidase/hypoxanthine-catalyzed nitroreduction of 2,7-diNF, 2-nitrofluorene (2-NF), and 1-nitropyrene (1-NP) yielded the respective amines to similar extents (30-50%). However, the level of the major adducts ( approximately 0.15/10(6) nucleotides) from 2-NF [N-(deoxyguanosin-8-yl)-2-aminofluorene] and 2,7-diNF [N-(deoxyguanosin-8-yl)-2-amino-7-NF] was 30 adducts/10(6) nucleotides, levels comparable to those from 1,6-dinitropyrene and 4- or 49-fold greater than the respective levels without acetyl CoA. Recovery of 2-nitroso-7-NF and 2-amino-7-NF from cytosol-catalyzed reduction of 2,7-diNF indicated nitroreduction and an N-hydroxy arylamine intermediate. Likewise, the presence of 2-acetylamino-7-NF indicated that reactivity with acyltransferase(s) was not prevented by the nitro group at C7. These data are consistent with activation of 2,7-diNF via nitroreduction to the N-hydroxy arylamine and acetyl CoA-dependent O-acetylation of the latter to bind to DNA. Enzymatic nitroreduction of 2,7-diNF was greatly enhanced by 9-oxidation. The nitroreduction of either 9-oxo-2,7-diNF or 9-hydroxy-2,7-diNF catalyzed by liver

  13. 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. Georg Thieme Verlag KG Stuttgart · New York.

  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. Copyright (c) 2009. Published by Elsevier Ireland Ltd.

  15. Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

    Science.gov (United States)

    2009-01-01

    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 ancestral gene that became

  16. Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds.

    Science.gov (United States)

    Kim, Hae Jin; Silva, Jillian E; Vu, Hieu Sy; Mockaitis, Keithanne; Nam, Jeong-Won; Cahoon, Edgar B

    2015-07-01

    Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8-C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina β-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  17. Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

    Science.gov (United States)

    Silverman, Andrew M; Qiao, Kangjian; Xu, Peng; Stephanopoulos, Gregory

    2016-04-01

    Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH

  18. Reduction of Ether-Type Glycerophospholipids, Plasmalogens, by NF-κB Signal Leading to Microglial Activation.

    Science.gov (United States)

    Hossain, Md Shamim; Abe, Yuichi; Ali, Fatma; Youssef, Mohammed; Honsho, Masanori; Fujiki, Yukio; Katafuchi, Toshihiko

    2017-04-12

    Neuroinflammation characterized by activation of glial cells is observed in various neurodegenerative diseases including Alzheimer's disease (AD). Although the reduction of ether-type glycerophospholipids, plasmalogens (Pls), in the brain is reported in AD patients, the mechanism of the reduction and its impact on neuroinflammation remained elusive. In the present study, we found for the first time that various inflammatory stimuli reduced Pls levels in murine glial cells via NF-κB activation, which then downregulated a Pls-synthesizing enzyme, glycerone phosphate O-acyltransferase (Gnpat) through increased c-Myc recruitment onto the Gnpat promoter. We also found that systemic injection of lipopolysaccharide, aging, and chronic restraint stress reduced brain Pls contents that were associated with glial NF-κB activation, an increase in c-Myc expression, and downregulation of Gnpat in the mouse cortex and hippocampus. More interestingly, the reduction of Pls contents in the murine cortex itself could increase the activated phenotype of microglial cells and the expression of proinflammatory cytokines, suggesting further acceleration of neuroinflammation by reduction of brain Pls. A similar mechanism of Gnpat reduction was also found in human cell lines, triple-transgenic AD mouse brain, and postmortem human AD brain tissues. These findings suggest a novel mechanism of neuroinflammation that may explain prolonged progression of AD and help us to explore preventive and therapeutic strategies to treat neurodegenerative diseases.SIGNIFICANCE STATEMENT Ether-type glycerophospholipids, plasmalogens (Pls), are reduced in the brain of Alzheimer disease (AD) patients. We found that inflammatory stimuli reduced Pls contents by downregulation of the Pls-synthesizing enzyme glycerone phosphate O-acyltransferase (Gnpat) through NF-κB-mediated recruitment of c-Myc onto the Gnpat promoter in both murine and human cell lines. Murine brains after systemic lipopolysaccharide

  19. Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds

    Science.gov (United States)

    Kim, Hae Jin; Silva, Jillian E.; Vu, Hieu Sy; Mockaitis, Keithanne; Nam, Jeong-Won; Cahoon, Edgar B.

    2015-01-01

    Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0–14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8–C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina β-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids. PMID:25969557

  20. Mining the bitter melon (momordica charantia l. seed transcriptome by 454 analysis of non-normalized and normalized cDNA populations for conjugated fatty acid metabolism-related genes

    Directory of Open Access Journals (Sweden)

    Shipp Matthew J

    2010-11-01

    Full Text Available Abstract Background Seeds of Momordica charantia (bitter melon produce high levels of eleostearic acid, an unusual conjugated fatty acid with industrial value. Deep sequencing of non-normalized and normalized cDNAs from developing bitter melon seeds was conducted to uncover key genes required for biotechnological transfer of conjugated fatty acid production to existing oilseed crops. It is expected that these studies will also provide basic information regarding the metabolism of other high-value novel fatty acids. Results Deep sequencing using 454 technology with non-normalized and normalized cDNA libraries prepared from bitter melon seeds at 18 DAP resulted in the identification of transcripts for the vast majority of known genes involved in fatty acid and triacylglycerol biosynthesis. The non-normalized library provided a transcriptome profile of the early stage in seed development that highlighted the abundance of transcripts for genes encoding seed storage proteins as well as for a number of genes for lipid metabolism-associated polypeptides, including Δ12 oleic acid desaturases and fatty acid conjugases, class 3 lipases, acyl-carrier protein, and acyl-CoA binding protein. Normalization of cDNA by use of a duplex-specific nuclease method not only increased the overall discovery of genes from developing bitter melon seeds, but also resulted in the identification of 345 contigs with homology to 189 known lipid genes in Arabidopsis. These included candidate genes for eleostearic acid metabolism such as diacylglycerol acyltransferase 1 and 2, and a phospholipid:diacylglycerol acyltransferase 1-related enzyme. Transcripts were also identified for a novel FAD2 gene encoding a functional Δ12 oleic acid desaturase with potential implications for eleostearic acid biosynthesis. Conclusions 454 deep sequencing, particularly with normalized cDNA populations, was an effective method for mining of genes associated with eleostearic acid metabolism in

  1. Comparative effect of fish oil feeding and other dietary fatty acids on plasma lipoproteins, biliary lipids, and hepatic expression of proteins involved in reverse cholesterol transport in the rat.

    Science.gov (United States)

    Morgado, Nora; Rigotti, Attilio; Valenzuela, Alfonso

    2005-01-01

    While elevated plasma high-density lipoprotein (HDL) levels has been associated to a reduction in cardiovascular risk, dietary fish oils rich in omega-3 polyunsaturated fatty acids (PUFAs) may protect against this disease. The protective effect of HDL is associated to its participation in the reverse cholesterol transport pathway. On the other hand, omega-3 PUFAs decrease plasma HDL levels compared to other fatty acids, which may suggest an effect on reverse cholesterol transport. In this work, the effect of dietary fish oil on the fatty acid composition of hepatic membranes, plasma lipoprotein cholesterol profile, biliary lipids, and the expression of proteins involved in reverse cholesterol transport, was compared to other dietary oils having a different degree of fatty acid unsaturation. Male rats were fed a semi synthetic diet containing fish oil (omega-3), sunflower oil (omega-6), olive oil (omega-9) or coconut oil (saturated). Hepatic membrane fatty acid composition, plasma cholesterol levels, lipoprotein cholesterol profile, biliary lipids, hepatic mRNA levels for lecithin cholesterol acyltransferase, hepatic lipase, apo E, and apo A-I, and hepatic protein levels of the scavenger receptor class B type I, caveolin-1, and the ATP binding cassette transporter A1 were analyzed. Plasma apo A-I and apo E protein levels were also evaluated. Compared to the other diets, omega-3 PUFAs significantly changed omega-3/omega-6 fatty acid ratio of hepatic membranes, caused a reduction of plasma total and HDL cholesterol, and selectively increased biliary cholesterol secretion. No modification in the expression levels of lecithin cholesterol acyltransferase, hepatic lipase, apo A-I and apo E mRNA was observed. Hepatic scavenger receptor class B type I, caveolin-1, and the ATP binding cassette transporter A1 protein levels were also not affected. Plasma apo A-I, but not apo E, was reduced. These results show that dietary omega-3 PUFAs reduce plasma HDL cholesterol and

  2. Increasing fatty acid oxidation remodels the hypothalamic neurometabolome to mitigate stress and inflammation.

    Directory of Open Access Journals (Sweden)

    Joseph W McFadden

    Full Text Available Modification of hypothalamic fatty acid (FA metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1 and fatty acid oxidation (FAOx, exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS, and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.

  3. Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Jauhiainen, Matti; Moser, Markus

    2008-01-01

    To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fold...... of alpha- to pre-alpha-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-beta-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 +/- 1.1%, p = 0.06) and decreased in apoM(-/-) mice (0.5 +/- 0.3%, p = 0.......03) versus controls (1.8 +/- 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with approximately 2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic...

  4. ACAT1 deficiency increases cholesterol synthesis in mouse peritoneal macrophages.

    Science.gov (United States)

    Dove, Dwayne E; Su, Yan Ru; Swift, Larry L; Linton, MacRae F; Fazio, Sergio

    2006-06-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies free cholesterol and stores cholesteryl esters in lipid droplets. Macrophage ACAT1 deficiency results in increased atherosclerotic lesion area in hyperlipidemic mice via disrupted cholesterol efflux, increased lipoprotein uptake, accumulation of intracellular vesicles, and accelerated apoptosis. The objective of this study was to determine whether lipid synthesis is affected by ACAT1. The synthesis, esterification, and efflux of new cholesterol were measured in peritoneal macrophages from ACAT1(-/-) mice. Cholesterol synthesis was increased by 134% (p=0.001) in ACAT1(-/-) macrophages compared to wildtype macrophages. Increased synthesis resulted in a proportional increase in the efflux of newly synthesized cholesterol. Although the esterification of new cholesterol was reduced by 93% (pSREBP1a mRNA was increased 6-fold in ACAT1(-/-) macrophages compared to wildtype macrophages, suggesting an up-regulation of cholesterol and fatty acid synthesis in ACAT1(-/-) macrophages. Increased cholesterol synthesis and up-regulation of SREBP in ACAT1(-/-) macrophages suggests that ACAT1 affects the regulation of lipid metabolism in macrophages. This change in cholesterol homeostasis may contribute to the atherogenic potential of ACAT1(-/-) macrophages.

  5. Moringa Leaves Prevent Hepatic Lipid Accumulation and Inflammation in Guinea Pigs by Reducing the Expression of Genes Involved in Lipid Metabolism

    Directory of Open Access Journals (Sweden)

    Manal Mused Almatrafi

    2017-06-01

    Full Text Available To investigate the mechanisms by which Moringa oleifera leaves (ML modulate hepatic lipids, guinea pigs were allocated to either control (0% ML, 10% Low Moringa (LM or 15% High Moringa (HM diets with 0.25% dietary cholesterol to induce hepatic steatosis. After 6 weeks, guinea pigs were sacrificed and liver and plasma were collected to determine plasma lipids, hepatic lipids, cytokines and the expression of genes involved in hepatic cholesterol (CH and triglyceride (TG metabolism. There were no differences in plasma lipids among groups. A dose-response effect of ML was observed in hepatic lipids (CH and TG with the lowest concentrations in the HM group (p < 0.001, consistent with histological evaluation of lipid droplets. Hepatic gene expression of diglyceride acyltransferase-2 and peroxisome proliferator activated receptor-γ, as well as protein concentrations interleukin (IL-1β and interferon-γ, were lowest in the HM group (p < 0.005. Hepatic gene expression of cluster of differentiation-68 and sterol regulatory element binding protein-1c were 60% lower in both the LM and HM groups compared to controls (p < 0.01. This study demonstrates that ML may prevent hepatic steatosis by affecting gene expression related to hepatic lipids synthesis resulting in lower concentrations of cholesterol and triglycerides and reduced inflammation in the liver.

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

    Science.gov (United States)

    Lin, Yuguang; 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 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. PMID:19228775

  7. Detection of molecular paths associated with insulitis and type 1 diabetes in non-obese diabetic mouse.

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    Erno Lindfors

    Full Text Available Recent clinical evidence suggests important role of lipid and amino acid metabolism in early pre-autoimmune stages of type 1 diabetes pathogenesis. We study the molecular paths associated with the incidence of insulitis and type 1 diabetes in the Non-Obese Diabetic (NOD mouse model using available gene expression data from the pancreatic tissue from young pre-diabetic mice. We apply a graph-theoretic approach by using a modified color coding algorithm to detect optimal molecular paths associated with specific phenotypes in an integrated biological network encompassing heterogeneous interaction data types. In agreement with our recent clinical findings, we identified a path downregulated in early insulitis involving dihydroxyacetone phosphate acyltransferase (DHAPAT, a key regulator of ether phospholipid synthesis. The pathway involving serine/threonine-protein phosphatase (PP2A, an upstream regulator of lipid metabolism and insulin secretion, was found upregulated in early insulitis. Our findings provide further evidence for an important role of lipid metabolism in early stages of type 1 diabetes pathogenesis, as well as suggest that such dysregulation of lipids and related increased oxidative stress can be tracked to beta cells.

  8. 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.

  9. 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.

  10. Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis.

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    Holger Jenke-Kodama

    2006-10-01

    Full Text Available Modular polyketide synthases (PKSs of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT, ketoreductase (KR, and dehydratase (DH-KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.

  11. Neutral lipid metabolism influences phospholipid synthesis and deacylation in Saccharomyces cerevisiae.

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    Gabriel Mora

    Full Text Available Establishment and maintenance of equilibrium in the fatty acid (FA composition of phospholipids (PL requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one.

  12. Pathophysilogical mechanism and treatment strategies for Leber congenital amaurosis.

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    Fu, Yingbin; Zhang, Tao

    2014-01-01

    Mutations in retinoid isomerase, RPE65, or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal recycling and cause Leber congenital amaurosis (LCA), the most severe retinal dystrophy in early childhood. We used Lrat (-/-), a murine model for LCA, to investigate the mechanism of rapid cone degeneration. We found that mislocalized M-opsin was degraded whereas mislocalized S-opsin accumulated in Lrat (-/-) cones before the onset of massive ventral/central cone degeneration. Since the ventral and central retina expresses higher levels of S-opsin than the dorsal retina in mice, our results may explain why ventral and central cones degenerate more rapidly than dorsal cones in Rpe65 (-/-) and Lrat (-/-) LCA models. In addition, human blue opsin and mouse S-opsin, but not mouse M-opsin or human red/green opsins, aggregated to form cytoplasmic inclusions in transfected cells, which may explain why blue cone function is lost earlier than red/green-cone function in LCA patients. The aggregation of short-wavelength opsins likely caused rapid cone degenerations through an ER stress pathway as demonstrated in both the Lrat (-/-) retina and transfected cells. Based on this mechanism, we designed a new therapy of LCA by reducing ER stress. We found that systemic injection of an ER chemical chaperone, tauroursodeoxycholic acid (TUDCA), is effective in reducing ER stress, preventing apoptosis, and preserving cones in Lrat (-/-) mice.

  13. Adrenal Function in females with low plasma HDL-C due to mutations in ABCA1 and LCAT.

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    Andrea E Bochem

    Full Text Available INTRODUCTION: Adrenal steroidogenesis is essential for human survival and depends on the availability of the precursor cholesterol. Male subjects with low plasma levels of high density lipoprotein (HDL cholesterol are characterized by decreased adrenal function. Whether this is also the case in female subjects with low plasma HDL-C levels is unresolved to date. FINDINGS: 15 female ATP binding cassette transporter AI (ABCAI and 14 female lecithin-cholesterol acyltransferase (LCAT were included in the study. HDL-C levels were 38% and 41% lower in ABCA1 and LCAT mutation carriers compared to controls, respectively. Urinary steroid excretion of 17-ketogenic steroids or 17-hydroxy corticosteroids did not differ between 15 female ABCA1 mutation carriers (p = 0.27 vs 0.30 respectively and 30 matched normolipidemic controls or between 14 female LCAT mutation carriers and 28 matched normolipidemic controls (p = 0.10 and 0.14, respectively. Cosyntropin testing in an unselected subgroup of 8 ABCA1 mutation carriers and 3 LCAT mutation carriers did not reveal differences between carriers and controls. CONCLUSION: Adrenal function in females with molecularly defined low HDL-C levels is not different from controls. The discrepancy with the finding of impaired steroidogenesis in males with molecularly defined low HDL-C levels underscores the importance of gender specific analyses in cholesterol-related research.

  14. Downregulation of the sodium channel Nav1.6 by potential transcriptomic deregulation may explain sensory deficits in critical illness neuropathy.

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    Li, Nan; Liu, Zhongmin; Wang, Guang; Wang, Shiji

    2015-12-15

    Sepsis patients and other patients in the critical care settings are at very high risk of mortality due to the primary illness. However, a fraction of patients, even after showing initial clinical improvement, deteriorates relentlessly at later stages. Increasingly, it is being identified that this is mostly due to dysfunction of the neurological system. We obtained peripheral nerve biopsies from the sural nerve from ICU patients. Nav1.6 expression was significantly diminished. The expression of cellular membrane anchoring protein for Nav1.6, ankyrin, remained unaffected, suggesting that genomic repression may be responsible for the diminished expression of the sodium channels. We examined the expression of two regulatory transcription factors: (a) a positive regulator YY1 that binds to the promoter region of sodium channels and (b) an upstream negative neuronal regulator REST. REST expression was significantly elevated, while YY1 expression was diminished. Finally, we also observed that the cholinergic synthetic enzyme acyltransferase was also significantly diminished in sensory nerve lysates. Finally, circulating antibodies was detected in the peripheral blood against all the major sodium channels Nav1.6, 1.8 and 1.9, which contribute to the development and propagation of action potentials. This may potentially explain why its dysfunction affects neurological functions across all systems of the body during critical illness. The underlying mechanism of why the expression of the REST transcriptional factor is affected in critical illnesses remains our future goals of investigation. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Very low levels of HDL cholesterol and atherosclerosis, a variable relationship--a review of LCAT deficiency.

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    Savel, Julia; Lafitte, Marianne; Pucheu, Yann; Pradeau, Vincent; Tabarin, Antoine; Couffinhal, Thierry

    2012-01-01

    A number of epidemiological and clinical studies have demonstrated that plasma high-density lipoprotein (HDL) level is a strong inverse predictor of cardiovascular events. HDL is believed to retard the formation of atherosclerotic lesions by removing excess cholesterol from cells and preventing endothelial dysfunction. Lecithin cholesterol acyltransferase (LCAT) plays a central role in the formation and maturation of HDL, and in the intravascular stage of reverse cholesterol transport: a major mechanism by which HDL modulates the development and progression of atherosclerosis. A defect in LCAT function would be expected to enhance atherosclerosis, by interfering with the reverse cholesterol transport step. As such, one would expect to find more atherosclerosis and cardiovascular events in LCAT-deficient patients. But this relationship is not always evident. In this review, we describe contradictory reports in the literature about cardiovascular risks in this patient population. We discuss the paradoxical finding of severe HDL deficiency and an absence of subclinical atherosclerosis in LCAT-deficient patients, which has been used to reject the hypothesis that HDL level is important in the protection against atherosclerosis. Furthermore, to illustrate this paradoxical finding, we present a case study of one patient, referred for evaluation of global cardiovascular risk in the presence of a low HDL cholesterol level, who was diagnosed with LCAT gene mutations.

  16. Very low levels of HDL cholesterol and atherosclerosis, a variable relationship – a review of LCAT deficiency

    Science.gov (United States)

    Savel, Julia; Lafitte, Marianne; Pucheu, Yann; Pradeau, Vincent; Tabarin, Antoine; Couffinhal, Thierry

    2012-01-01

    A number of epidemiological and clinical studies have demonstrated that plasma high-density lipoprotein (HDL) level is a strong inverse predictor of cardiovascular events. HDL is believed to retard the formation of atherosclerotic lesions by removing excess cholesterol from cells and preventing endothelial dysfunction. Lecithin cholesterol acyltransferase (LCAT) plays a central role in the formation and maturation of HDL, and in the intravascular stage of reverse cholesterol transport: a major mechanism by which HDL modulates the development and progression of atherosclerosis. A defect in LCAT function would be expected to enhance atherosclerosis, by interfering with the reverse cholesterol transport step. As such, one would expect to find more atherosclerosis and cardiovascular events in LCAT-deficient patients. But this relationship is not always evident. In this review, we describe contradictory reports in the literature about cardiovascular risks in this patient population. We discuss the paradoxical finding of severe HDL deficiency and an absence of subclinical atherosclerosis in LCAT-deficient patients, which has been used to reject the hypothesis that HDL level is important in the protection against atherosclerosis. Furthermore, to illustrate this paradoxical finding, we present a case study of one patient, referred for evaluation of global cardiovascular risk in the presence of a low HDL cholesterol level, who was diagnosed with LCAT gene mutations. PMID:22701329

  17. Organization of human ACAT-2 gene and its cell-type-specific promoter activity.

    Science.gov (United States)

    Song, B L; Qi, W; Yang, X Y; Chang, C C; Zhu, J Q; Chang, T Y; Li, B L

    2001-03-30

    Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Two ACAT genes exist in mammals. We report here the genomic organization of human ACAT-2 gene and analysis of its promoter activity in various cell lines. The human ACAT-2 gene spans over 18 kb and contains 15 exons. Three transcription start sites and one poly(A) site are identified by the 5'/3'-RACE. In addition, the human ACAT-2 gene is linked to the insulin-like growth factor binding protein 6 (IGFBP-6) gene in a head-to-tail manner with a small intergenic region of about 1.2 kb. The 5'-flanking region of human ACAT-2 gene contains many potential cis-acting elements for multiple transcriptional regulatory factors but lacks TATA and CCAAT boxes. Using promoter-luciferase reporter assays, we demonstrate the transcriptional activity of ACAT-2 gene promoter is high in Caco-2 cells, especially after these cells become postconfluent and behave as intestinal enterocytes. Copyright 2001 Academic Press.

  18. ACAT inhibition reduces the progression of preexisting, advanced atherosclerotic mouse lesions without plaque or systemic toxicity.

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    Rong, James X; Blachford, Courtney; Feig, Jonathan E; Bander, Ilda; Mayne, Jeffrey; Kusunoki, Jun; Miller, Christine; Davis, Matthew; Wilson, Martha; Dehn, Shirley; Thorp, Edward; Tabas, Ira; Taubman, Mark B; Rudel, Lawrence L; Fisher, Edward A

    2013-01-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice. Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.

  19. ACAT inhibitory activity of exudates from Calocedrus macrolepis var. formosana.

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    Hsieh, Yu-Hsin; Chen, Kuan-Jung; Chien, Shih-Chang; Cheng, Wen-Ling; Xiao, Jun-Hong; Wang, Sheng-Yang

    2012-12-01

    Cholesterol acyltransferase (ACAT) is an enzyme controlling cholesterol esterification in cells. Large amounts of cholesterol esters accumulate in macrophages and smooth muscle cells of blood vessel walls resulting in the initial stages of atherosclerosis. Thus, atherosclerosis might be inhibited through inhibition of the activity of ACAT. In the present study, we identified by spectral analysis and chromatographic quantification that ferruginol was the most abundant component of exudates secreted from the wounding site of Calocedrus macrolepis Kurz var. formosana. Results obtained from the cholesterol absorption assay revealed that ferruginol exhibited a significant inhibitory activity on cholesterol absorption in mice macrophages (RAW 264.7 cell). Based on the results from analyzing the ratio of cholesterol esterification, ferruginol dose-dependently suppressed cholesterol esterification and the IC50 value was 2.0 microg/mL. In conclusion, ferruginol revealed strong inhibitory activities that retarded the absorption and esterification of cholesterol in cells. Our finding indicates that ferruginol might possess a potential for development as a pharmaceutical product for preventing arteriosclerosis.

  20. Effect of VULM 1457, an ACAT inhibitor, on serum lipid levels and on real time red blood cell flow in diabetic and non-diabetic hamsters fed high cholesterol-lipid diet.

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    Vojtassáková, E; Syneková, M; Tazká, D; Mátyás, S; Hózová, R; Sadlonová, I; Svec, P

    2007-12-01

    Acyl-coenzyme A: cholesterol O-acyltransferase (ACAT) catalyzes the formation of cholesterol/fatty acyl-coenzyme A esters. Accumulation of cholesterol esters leads to pathological changes connected with atherosclerosis. We have evaluated effects of a newly synthesized ACAT inhibitor, 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM 1457), on serum lipid (cholesterol and triglycerides) levels and velocity of red blood cells (RBC) in non-diabetic and diabetic hamsters fed on high cholesterol-lipid (HCHL) diet during 3 months. The VULM 1457 effects on the paw microcirculation were assessed using capillary microscopy by measuring (RBC) velocity in vivo. Hamsters fed on HCHL diet became hypercholesterolemic with a dramatic increase in serum lipids accompanied with significantly decreased RBC velocity. Diabetic hamsters fed on HCHL diet had further increased serum lipids with reduction of RBC velocity. The VULM 1457 inhibitor lowered cholesterol levels in both non-diabetic and diabetic hamsters fed on HCHL diet. The greater VULM 1457 effect was shown in diabetic hamsters fed on HCHL diet where VULM 1457 expressed hypotriglycerides effects, too. An improved RBC velocity-pronounced effect was observed in diabetic hamsters fed on HCHL diet treated with VULM 1457. These results suggest that the ACAT inhibitor, VULM 1457, is a prospective hypolipidemic and anti-atherogenic drug which treats diabetes.

  1. Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

    Science.gov (United States)

    Smith, Jeffery L; Rangaraj, Kavitha; Simpson, Robert; Maclean, Donald J; Nathanson, Les K; Stuart, Katherine A; Scott, Shaun P; Ramm, Grant A; de Jersey, John

    2004-04-01

    ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

  2. Esculeogenin A, a new tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in ApoE-deficient mice by inhibiting ACAT.

    Science.gov (United States)

    Fujiwara, Yukio; Kiyota, Naoko; Hori, Masaharu; Matsushita, Sayaka; Iijima, Yoko; Aoki, Koh; Shibata, Daisuke; Takeya, Motohiro; Ikeda, Tsuyoshi; Nohara, Toshihiro; Nagai, Ryoji

    2007-11-01

    We recently identified esculeoside A, a new spirosolane-type glycoside, with a content in tomatoes that is 4-fold higher than that of lycopene. In the present study, we examined the effects of esculeoside A and esculeogenin A, a new aglycon of esculeoside A, on foam cell formation in vitro and atherogenesis in apoE-deficient mice. Esculeogenin A significantly inhibited the accumulation of cholesterol ester (CE) induced by acetylated low density lipoprotein (acetyl-LDL) in human monocyte-derived macrophages (HMDM) in a dose-dependent manner without inhibiting triglyceride accumulation, however, it did not inhibit the association of acetyl-LDL to the cells. Esculeogenin A also inhibited CE formation in Chinese hamster ovary cells overexpressing acyl-coenzymeA (CoA): cholesterol acyl-transferase (ACAT)-1 or ACAT-2, suggesting that esculeogenin A suppresses the activity of both ACAT-1 and ACAT-2. Furthermore, esculeogenin A prevented the expression of ACAT-1 protein, whereas that of SR-A and SR-BI was not suppressed. Oral administration of esculeoside A to apoE-deficient mice significantly reduced the levels of serum cholesterol, triglycerides, LDL-cholesterol, and the areas of atherosclerotic lesions without any detectable side effects. Our study provides the first evidence that purified esculeogenin A significantly suppresses the activity of ACAT protein and leads to reduction of atherogenesis.

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

    Science.gov (United States)

    Lin, Yuguang; 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 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.

  4. Novel N-terminal cleavage of APP precludes Abeta generation in ACAT-defective AC29 cells.

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    Huttunen, Henri J; Puglielli, Luigi; Ellis, Blake C; MacKenzie Ingano, Laura A; Kovacs, Dora M

    2009-01-01

    A common pathogenic event that occurs in all forms of Alzheimer's disease is the progressive accumulation of amyloid beta-peptide (Abeta) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Abeta from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both alpha and the amyloidogenic beta cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Abeta generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Abeta generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment.

  5. Association between Single Nucleotide Polymorphism rs1044925 and the Risk of Coronary Artery Disease and Ischemic Stroke

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    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.

  6. Identification of ACAT1- and ACAT2-specific inhibitors using a novel, cell-based fluorescence assay: individual ACAT uniqueness.

    Science.gov (United States)

    Lada, Aaron T; Davis, Matthew; Kent, Carol; Chapman, James; Tomoda, Hiroshi; Omura, Satoshi; Rudel, Lawrence L

    2004-02-01

    Acyl CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2 are enzymes responsible for the formation of cholesteryl esters in tissues. While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. In this study, NBD-cholesterol was used to screen for specific inhibitors of ACAT1 and ACAT2. Incubation of AC29 cells, which do not contain ACAT activity, with NBD-cholesterol showed weak fluorescence when the compound was localized in the membrane. When AC29 cells stably transfected with either ACAT1 or ACAT2 were incubated with NBD-cholesterol, the fluorescent signal localized to the nonpolar core of cytoplasmic lipid droplets was strongly fluorescent and was correlated with two independent measures of ACAT activity. Several compounds were found to have greater inhibitory activity toward ACAT1 than ACAT2, and one compound was identified that specifically inhibits ACAT2. The demonstration of selective inhibition of ACAT1 and ACAT2 provides evidence for uniqueness in structure and function of these two enzymes. To the extent that ACAT2 is confined to hepatocytes and enterocytes, the only two cell types that secrete lipoproteins, selective inhibition of ACAT2 may prove to be most beneficial in the reduction of plasma lipoprotein cholesterol concentrations.

  7. An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2) and their proposed role in metabolic processes in pig.

    Science.gov (United States)

    Sodhi, Simrinder Singh; Ghosh, Mrinmoy; Song, Ki Duk; Sharma, Neelesh; Kim, Jeong Hyun; Kim, Nam Eun; Lee, Sung Jin; Kang, Chul Woong; Oh, Sung Jong; Jeong, Dong Kee

    2014-01-01

    The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (PACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

  8. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.

    Science.gov (United States)

    Yagyu, H; Kitamine, T; Osuga, J; Tozawa, R; Chen, Z; Kaji, Y; Oka, T; Perrey, S; Tamura, Y; Ohashi, K; Okazaki, H; Yahagi, N; Shionoiri, F; Iizuka, Y; Harada, K; Shimano, H; Yamashita, H; Gotoda, T; Yamada, N; Ishibashi, S

    2000-07-14

    Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes esterification of cellular cholesterol. To investigate the role of ACAT-1 in atherosclerosis, we have generated ACAT-1 null (ACAT-1-/-) mice. ACAT activities were present in the liver and intestine but were completely absent in adrenal, testes, ovaries, and peritoneal macrophages in our ACAT-1-/- mice. The ACAT-1-/- mice had decreased openings of the eyes because of atrophy of the meibomian glands, a modified form of sebaceous glands normally expressing high ACAT activities. This phenotype is similar to dry eye syndrome in humans. To determine the role of ACAT-1 in atherogenesis, we crossed the ACAT-1-/- mice with mice lacking apolipoprotein (apo) E or the low density lipoprotein receptor (LDLR), hyperlipidemic models susceptible to atherosclerosis. High fat feeding resulted in extensive cutaneous xanthomatosis with loss of hair in both ACAT-1-/-:apo E-/- and ACAT-1-/-:LDLR-/- mice. Free cholesterol content was significantly increased in their skin. Aortic fatty streak lesion size as well as cholesteryl ester content were moderately reduced in both double mutant mice compared with their respective controls. These results indicate that the local inhibition of ACAT activity in tissue macrophages is protective against cholesteryl ester accumulation but causes cutaneous xanthomatosis in mice that lack apo E or LDLR.

  9. An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2 and their proposed role in metabolic processes in pig.

    Directory of Open Access Journals (Sweden)

    Simrinder Singh Sodhi

    Full Text Available The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2 is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2 was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V and 281 (R to H were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05. Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

  10. Sex-specific association of ACAT-1 rs1044925 SNP and serum lipid levels in the hypercholesterolemic subjects.

    Science.gov (United States)

    Wu, Dong-Feng; Yin, Rui-Xing; Aung, Lynn Htet Htet; Li, Qing; Yan, Ting-Ting; Zeng, Xiao-Na; Huang, Ke-Ke; Huang, Ping; Wu, Jin-Zhen; Pan, Shang-Ling

    2012-01-13

    Acyl-CoA:cholesterol acyltransferase (ACAT) is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. The cellular cholesterol efflux correlated with serum high-density lipoprotein cholesterol (HDL-C) concentrations has shown to be impaired in hyperlipidemic mice. The present study was carried out to clarify the association of ACAT-1 rs1044925 single nucleotide polymorphism (SNP) and serum lipid levels in the hyperlipidemic subjects. A total of 821 unrelated subjects (hyperlipidemia, 476; normolipidemia, 345) aged 15-80 were included in the study. Genotyping of the ACAT-1 rs1044925 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. There was no significant difference in the genotypic and allelic frequencies of ACAT-1 rs1044925 SNP between the normolipidemic and hyperlipidemic subjects. The levels of total cholesterol (TC), HDL-C and apolipoprotein (Apo) AI in hyperlipidemic subjects were different between the AA and AC/CC genotypes in male but not in female (P ACAT-1 rs1044925 SNP in male hyperlipidemic subjects had higher serum TC, HDL-C and ApoAI levels than the C allele noncarriers. There is a sex (male)-specific association of ACAT-1 rs1044925 SNP and serum HDL-C and ApoAI levels in the hypercholesterolemic subjects.

  11. Absolute stereochemistry of fungal beauveriolide III and ACAT inhibitory activity of four stereoisomers.

    Science.gov (United States)

    Ohshiro, Taichi; Namatame, Ichiji; Nagai, Kenichiro; Sekiguchi, Takafumi; Doi, Takayuki; Takahashi, Takashi; Akasaka, Kazuaki; Rudel, Lawrence L; Tomoda, Hiroshi; Omura, Satoshi

    2006-09-29

    Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII. Furthermore, the four beauveriolide III isomers ((3S,4S)BeauIII (23a), (3R,4R)BeauIII (23b), (3R,4S)BeauIII (23c), and (3S,4R)BeauIII (23d)) were synthesized, and it was shown that all the spectral data for 23a were identical with those for natural 1b. Isomers 23a and 23d showed potent inhibitory activity of lipid droplet accumulation in macrophages, while the other two isomers caused weak inhibition. Thus, the 3S configuration of BeauIII is important for this activity. Furthermore, 23a and 23d showed rather specific inhibition against the ACAT1 isozyme.

  12. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels.

    Science.gov (United States)

    Ikenoya, Mami; Yoshinaka, Yasunobu; Kobayashi, Hideyuki; Kawamine, Katsumi; Shibuya, Kimiyuki; Sato, Fumiyasu; Sawanobori, Kimio; Watanabe, Takuya; Miyazaki, Akira

    2007-04-01

    Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1), a major ACAT isozyme in macrophages, plays an essential role in foam cell formation in atherosclerotic lesions. However, whether pharmacological inhibition of macrophage ACAT-1 causes exacerbation or suppression of atherosclerosis is controversial. We developed and characterized a novel ACAT inhibitor, K-604. The IC(50) values of K-604 for human ACAT-1 and ACAT-2 were 0.45 and 102.85 micromol/L, respectively, indicating that K-604 is 229-fold more selective for ACAT-1. Kinetic analysis indicated that the inhibition was competitive with respect to oleoyl-coenzyme A with a K(i) value of 0.378 micromol/L. Exposure of human monocyte-derived macrophages to K-604 inhibited cholesterol esterification with IC(50) of 68.0 nmol/L. Furthermore, cholesterol efflux from THP-1 macrophages to HDL(3) or apolipoprotein A-I was enhanced by K-604. Interestingly, administration of K-604 to F1B hamsters on a high-fat diet at a dose of >or=1mg/kg suppressed fatty streak lesions without affecting plasma cholesterol levels. K-604, a potent and selective inhibitor of ACAT-1, suppressed the development of atherosclerosis in an animal model without affecting plasma cholesterol levels, providing direct evidence that pharmacological inhibition of ACAT-1 in the arterial walls leads to suppression of atherosclerosis.

  13. A selective ACAT-1 inhibitor, K-604, stimulates collagen production in cultured smooth muscle cells and alters plaque phenotype in apolipoprotein E-knockout mice.

    Science.gov (United States)

    Yoshinaka, Yasunobu; Shibata, Haruki; Kobayashi, Hideyuki; Kuriyama, Hiroki; Shibuya, Kimiyuki; Tanabe, Sohei; Watanabe, Takuya; Miyazaki, Akira

    2010-11-01

    Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1) plays an essential role in macrophage foam cell formation and progression of atherosclerosis. We developed a potent and selective ACAT-1 inhibitor, K-604, and tested its effects in apoE-knockout mice. Administration of K-604 to 8-week-old apoE-knockout mice for 12 weeks at a dose of 60 mg/kg/day significantly reduced macrophage-positive area and increased collagen-positive area in atherosclerotic plaques in the aorta without affecting plasma cholesterol levels or lesion areas, indicating direct plaque-modulating effects of K-604 on vascular walls independent of plasma cholesterol levels. Pactimibe, a nonselective inhibitor of ACAT-1 and ACAT-2, reduced plasma cholesterol levels but did not affect macrophage- or collagen-positive areas. The size of macrophages and cholesteryl ester contents in the aorta were reduced by K-604. Exposure of cultured human aortic smooth muscle cells to K-604 resulted in increased procollagen type 1 contents in the culture supernatant and increased procollagen type 1 mRNA levels. Procollagen production was unaffected by pactimibe even at a concentration that inhibited cholesterol esterification to the basal level. Thus, the plaque-modulating effects of K-604 can be explained by stimulation of procollagen production independent of ACAT inhibition in addition to potent inhibition of macrophage ACAT-1. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  14. Novel N-terminal Cleavage of APP Precludes Aβ Generation in ACAT-Defective AC29 Cells

    Science.gov (United States)

    Huttunen, Henri J.; Puglielli, Luigi; Ellis, Blake C.; Ingano, Laura A. MacKenzie

    2009-01-01

    A common pathogenic event that occurs in all forms of Alzheimer’s disease is the progressive accumulation of amyloid β-peptide (Aβ) in brain regions responsible for higher cognitive functions. Inhibition of acyl-coenzyme A: cholesterol acyltransferase (ACAT), which generates intracellular cholesteryl esters from free cholesterol and fatty acids, reduces the biogenesis of the Aβ from the amyloid precursor protein (APP). Here we have used AC29 cells, defective in ACAT activity, to show that ACAT activity steers APP either toward or away from a novel proteolytic pathway that replaces both α and the amyloidogenic β cleavages of APP. This alternative pathway involves a novel cleavage of APP holoprotein at Glu281, which correlates with reduced ACAT activity and Aβ generation in AC29 cells. This sterol-dependent cleavage of APP occurs in the endosomal compartment after internalization of cell surface APP. The resulting novel C-terminal fragment APP-C470 is destined to proteasomal degradation limiting the availability of APP for the Aβ generating system. The proportion of APP molecules that are directed to the novel cleavage pathway is regulated by the ratio of free cholesterol and cholesteryl esters in cells. These results suggest that subcellular cholesterol distribution may be an important regulator of the cellular fate of APP holoprotein and that there may exist several competing proteolytic systems responsible for APP processing within the endosomal compartment. PMID:18618086

  15. Cytotoxic and ACAT-inhibitory sesquiterpene lactones from the root of Ixeris dentata forma albiflora.

    Science.gov (United States)

    Ahn, Eun-Mi; Bang, Myun-Ho; Song, Myoung-Chong; Park, Mi-Hyun; Kim, Hwa-Young; Kwon, Byoung-Mog; Baek, Nam-In

    2006-11-01

    Ixeris dentata forma albiflora was extracted with 80% aqueous MeOH, and the concentrated extract was partitioned with EtOAc, n-BuOH and H2O. Eight sesquiterpenes were isolated through repeated silica gel and octadecyl silica gel (C18, ODS) column chromatography of the EtOAc and n-BuOH fractions. Physicochemical analysis using NMR, MS and IR revealed the chemical structures of the sesquiterpenes, which were zaluzanin (1), 9a-hydroxyguaian-4(15),10(14),11(13)-triene-6,12-olide (2), 3beta-O-beta-D-glucopyranosyl-8beta-hydroxyguaian-4(15),10(14)-diene-6,12-olide (3), 3-O-beta-D-glucopyranosyl-8beta-hydroxyguauan-10(14)-ene-6,12-olide (4), ixerin M (5), glucozaluzanin C (6), crepiside I (7), and ixerin D (8). This is the first time that these sesquiterpene lactones have been isolated from this plant. Compounds 1, 2 and 7 revealed relatively high cytotoxicities on human colon carcinoma cell and lung adenocarcinoma cell, while compounds 5 and 7 showed acyl-CoA: cholesterol acyltransferase (ACAT) inhibitory activity.

  16. In vitro metabolism of pyripyropene A and ACAT inhibitory activity of its metabolites.

    Science.gov (United States)

    Matsuda, Daisuke; Ohshiro, Taichi; Ohtawa, Masaki; Yamazaki, Hiroyuki; Nagamitsu, Tohru; Tomoda, Hiroshi

    2015-01-01

    Pyripyropene A (PPPA, 1) of fungal origin, a selective inhibitor of acyl-CoA:cholesterol acyltransferase 2 (ACAT2), proved orally active in atherogenic mouse models. The in vitro metabolites of 1 in liver microsomes and plasma of human, rabbit, rat and mouse were analyzed by ultra fast liquid chromatography and liquid chromatography/tandem mass spectrometry. In the liver microsomes from all species, successive hydrolysis occurred at the 1-O-acetyl residue, then at the 11-O-acetyl residue of 1, while the 7-O-acetyl residue was resistant to hydrolysis. Furthermore, dehydrogenation of the newly generated 11-alcoholic hydroxyl residue occurred in human and mouse-liver microsomes, while oxidation of the pyridine ring occurred in human and rabbit liver microsomes. On the other hand, hydrolysis of the 7-O-acetyl residue proceeded only in the mouse plasma. These data indicated that the in vitro metabolic profiles of 1 have subtle differences among animal species. All of the PPPA metabolites observed in liver microsomes and plasma markedly decreased ACAT2 inhibitory activity. These findings will help us to synthesize new PPPA derivatives more effective in in vivo study than 1.

  17. The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Hutter-Paier, Birgit; Huttunen, Henri J; Puglielli, Luigi; Eckman, Christopher B; Kim, Doo Yeon; Hofmeister, Alexander; Moir, Robert D; Domnitz, Sarah B; Frosch, Matthew P; Windisch, Manfred; Kovacs, Dora M

    2004-10-14

    Amyloid beta-peptide (Abeta) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD). We have previously reported that a well-characterized acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, inhibits Abeta production in cell-based experiments. Here, we assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.

  18. Sex-specific association of ACAT-1 rs1044925 SNP and serum lipid levels in the hypercholesterolemic subjects

    Directory of Open Access Journals (Sweden)

    Wu Dong-Feng

    2012-01-01

    Full Text Available Abstract Background Acyl-CoA:cholesterol acyltransferase (ACAT is a key enzyme in cellular cholesterol homeostasis and in atherosclerosis. The cellular cholesterol efflux correlated with serum high-density lipoprotein cholesterol (HDL-C concentrations has shown to be impaired in hyperlipidemic mice. The present study was carried out to clarify the association of ACAT-1 rs1044925 single nucleotide polymorphism (SNP and serum lipid levels in the hyperlipidemic subjects. Methods A total of 821 unrelated subjects (hyperlipidemia, 476; normolipidemia, 345 aged 15-80 were included in the study. Genotyping of the ACAT-1 rs1044925 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Results There was no significant difference in the genotypic and allelic frequencies of ACAT-1 rs1044925 SNP between the normolipidemic and hyperlipidemic subjects. The levels of total cholesterol (TC, HDL-C and apolipoprotein (Apo AI in hyperlipidemic subjects were different between the AA and AC/CC genotypes in male but not in female (P Conclusions The present study shows that the C allele carriers of ACAT-1 rs1044925 SNP in male hyperlipidemic subjects had higher serum TC, HDL-C and ApoAI levels than the C allele noncarriers. There is a sex (male-specific association of ACAT-1 rs1044925 SNP and serum HDL-C and ApoAI levels in the hypercholesterolemic subjects.

  19. The ACAT inhibitor avasimibe increases the fractional clearance rate of postprandial triglyceride-rich lipoproteins in miniature pigs.

    Science.gov (United States)

    Burnett, John R; Telford, Dawn E; Barrett, P Hugh R; Huff, Murray W

    2005-12-30

    Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n=5; 25 mg/kg/day, n=4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II. Avasimibe decreased the 2-h TRL (d20) triglyceride concentrations by 34%. The TRL triglyceride 0-12 h area under the curve (AUC) was decreased by 21%. In contrast, avasimibe had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma, however, the TRL RP 0-12 h AUC was decreased by 17%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate (FCR) was increased 1.4-fold with avasimibe. The TRL RP FCR was negatively correlated with very low density lipoprotein (VLDL) apoB production rate measured in the fasting state (r=-0.504). No significant changes in total intestinal lipid concentrations were observed. Thus, although avasimibe had no effect on intestinal TRL secretion, plasma TRL clearance was significantly increased; an effect that may relate to a decreased competition with hepatic VLDL for removal processes.

  20. ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane

    Science.gov (United States)

    Joyce, Charles W.; Shelness, Gregory S.; Davis, Matthew A.; Lee, Richard G.; Skinner, Kelly; Anderson, Richard A.; Rudel, Lawrence L.

    2000-01-01

    A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly. PMID:11071899

  1. Identification of potential ACAT-2 selective inhibitors using pharmacophore, SVM and SVR from Chinese herbs.

    Science.gov (United States)

    Qiao, Lian-Sheng; Zhang, Xian-Bao; Jiang, Lu-di; Zhang, Yan-Ling; Li, Gong-Yu

    2016-11-01

    Acyl-coenzyme A cholesterol acyltransferase (ACAT) plays an important role in maintaining cellular and organismal cholesterol homeostasis. Two types of ACAT isozymes with different functions exist in mammals, named ACAT-1 and ACAT-2. Numerous studies showed that ACAT-2 selective inhibitors are effective for the treatment of hypercholesterolemia and atherosclerosis. However, as a typical endoplasmic reticulum protein, ACAT-2 protein has not been purified and revealed, so combinatorial ligand-based methods might be the optimal strategy for discovering the ACAT-2 selective inhibitors. In this study, selective pharmacophore models of ACAT-1 inhibitors and ACAT-2 inhibitors were built, respectively. The optimal pharmacophore model for each subtype was identified and utilized as queries for the Traditional Chinese Medicine Database screening. A total of 180 potential ACAT-2 selective inhibitors were obtained, which were identified using an ACAT-2 pharmacophore and not by our ACAT-1 model. Selective SVM model and bioactive SVR model were generated for further identification of the obtained ACAT-2 inhibitors. Ten compounds were finally obtained with predicted inhibitory activities toward ACAT-2. Hydrogen bond acceptor, 2D autocorrelations, GETAWAY descriptors, and BCUT descriptors were identified as key structural features for selectivity and activity of ACAT-2 inhibitors. This study provides a reasonable ligand-based approach to discover potential ACAT-2 selective inhibitors from Chinese herbs, which could help in further screening and development of ACAT-2 selective inhibitors.

  2. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages.

    Science.gov (United States)

    Miyazaki, A; Sakashita, N; Lee, O; Takahashi, K; Horiuchi, S; Hakamata, H; Morganelli, P M; Chang, C C; Chang, T Y

    1998-10-01

    The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.

  3. ACAT inhibition reduces the progression of pre-existing, advanced atherosclerotic mouse lesions without plaque or systemic toxicity

    Science.gov (United States)

    Rong, James X.; Blachford, Courtney; Feig, Jonathan E.; Bander, Ilda; Mayne, Jeffrey; Kusunoki, Jun; Miller, Christine; Davis, Matthew; Wilson, Martha; Dehn, Shirley; Thorp, Edward; Tabas, Ira; Taubman, Mark B.; Rudel, Lawrence L.; Fisher, Edward A.

    2013-01-01

    Objective Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice due to cytotoxicity from free cholesterol accumulation, while we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on pre-established, advanced lesions of apoE-/- mice. Methods & Results ApoE-/- mice on Western diet for 14 weeks developed advanced plaques, and were either sacrificed (“Baseline”), or continued on Western diet without or with F1394 and sacrificed after 14 more weeks. F1394 was not associated with systemic toxicity. Compared to the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression, and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared to the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol, plaque necrosis was not increased, and efferocytosis (phagocytic clearance of apoptotic cells) was not impaired. The effects of F1394 were independent of changes in plasma cholesterol levels. Conclusions Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apoE-/- mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis in spite of recent trial data. PMID:23139293

  4. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

    Science.gov (United States)

    Chang, C C; Sakashita, N; Ornvold, K; Lee, O; Chang, E T; Dong, R; Lin, S; Lee, C Y; Strom, S C; Kashyap, R; Fung, J J; Farese, R V; Patoiseau, J F; Delhon, A; Chang, T Y

    2000-09-08

    By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

  5. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

    Science.gov (United States)

    Borrelli, Grazia M.; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  6. Structure of a Novel Enzyme That Catalyzes Acyl Transfer to Alcohols in Aqueous Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Mathews, I.; Soltis, M.; Saldajeno, M.; Ganshaw, G.; Sala, R.; Weyler, W.; Cervin, M.A.; Whited, G.; Bott, R.

    2009-06-03

    The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 {angstrom} resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.

  7. Kalpaamruthaa ameliorates mitochondrial and metabolic alterations in diabetes mellitus induced cardiovascular damage.

    Science.gov (United States)

    Latha, Raja; Shanthi, Palanivelu; Sachdanandam, Panchanadham

    2014-12-01

    Efficacy of Kalpaamruthaa on the activities of lipid and carbohydrate metabolic enzymes, electron transport chain complexes and mitochondrial ATPases were studied in heart and liver of experimental rats. Cardiovascular damage (CVD) was developed in 8 weeks after type 2 diabetes mellitus induction with high fat diet (2 weeks) and low dose of streptozotocin (2 × 35 mg/kg b.w. i.p. in 24 hr interval). In CVD-induced rats, the activities of total lipase, cholesterol ester hydrolase and cholesterol ester synthetase were increased, while lipoprotein lipase and lecithin-cholesterol acyltransferase activities were decreased. The activities of lipid-metabolizing enzymes were altered by Kalpaamruthaa in CVD-induced rats towards normal. Kalpaamruthaa modulated the activities of glycolytic enzymes (hexokinase, phosphogluco-isomerase, aldolase and glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1, 6-bisphosphatase) and glycogenolytic enzyme (glycogen phosphorylase) along with increased glycogen content in the liver of CVD-induced rats. The activities of isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, α-ketoglutarate dehydrogenase, Complexes and ATPases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase) were decreased in CVD-induced rats, which were ameliorated by the treatment with Kalpaamruthaa. This study ascertained the efficacy of Kalpaamruthaa for the treatment of CVD in diabetes through the modulation of metabolizing enzymes and mitochondrial dysfunction.

  8. 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

    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...... 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...

  9. Directed Evolution and Structural Characterization of a Simvastatin Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xue; Xie, Xinkai; Pashkov, Inna; Sawaya, Michael R.; Laidman, Janel; Zhang, Wenjun; Cacho, Ralph; Yeates, Todd O.; Tang, Yi; UCLA

    2010-02-02

    Enzymes from natural product biosynthetic pathways are attractive candidates for creating tailored biocatalysts to produce semisynthetic pharmaceutical compounds. LovD is an acyltransferase that converts the inactive monacolin J acid (MJA) into the cholesterol-lowering lovastatin. LovD can also synthesize the blockbuster drug simvastatin using MJA and a synthetic {alpha}-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD toward semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility, and thermal stability were obtained, with the best mutant displaying an {approx}11-fold increase in an Escherichia coli-based biocatalytic platform. To understand the structural basis of LovD enzymology, seven X-ray crystal structures were determined, including the parent LovD, an improved mutant G5, and G5 cocrystallized with ligands. Comparisons between the structures reveal that beneficial mutations stabilize the structure of G5 in a more compact conformation that is favorable for catalysis.

  10. Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism.

    Science.gov (United States)

    Yao, Lin; Tan, Chong; Song, Jinzhu; Yang, Qian; Yu, Lijie; Li, Xinling

    2016-01-01

    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 (5669bp) and pksT-2 (7901bp) 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. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  11. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication1[OPEN

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Ma, Biao; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Shui, Guang-Hou; Chen, Shou-Yi

    2017-01-01

    Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. PMID:28184009

  12. Expression of Castor LPAT2 Enhances Ricinoleic Acid Content at the sn-2 Position of Triacylglycerols in Lesquerella Seed

    Directory of Open Access Journals (Sweden)

    Grace Q. Chen

    2016-04-01

    Full Text Available Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA. Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and sn-3 positions of triacylglycerol (TAG, whereas castor contains ricinoleic acid (18:1OH at all three positions. To investigate whether lesquerella can be engineered to accumulate HFAs in the sn-2 position, multiple transgenic lines were made that express castor lysophosphatidic acid acyltransferase 2 (RcLPAT2 in the seed. RcLPAT2 increased 18:1OH at the sn-2 position of TAGs from 2% to 14%–17%, which resulted in an increase of tri-HFA-TAGs from 5% to 13%–14%. Our result is the first example of using a LPAT to increase ricinoleic acid at the sn-2 position of seed TAG. This work provides insights to the mechanism of HFA-containing TAG assembly in lesquerella and directs future research to optimize this plant for HFA production.

  13. Retinoids modulate thioacetamide-induced acute hepatotoxicity.

    Science.gov (United States)

    Shmarakov, Igor O; Borschovetska, Vira L; Marchenko, Mykhailo M; Blaner, William S

    2014-06-01

    The literature indicates that retinoids can influence the metabolism and actions of xenobiotics and conversely that xenobiotics can influence the metabolism and actions of retinoids. We were interested in understanding the degree to which hepatic retinoid stores, accumulated over a lifetime, affect xenobiotic metabolism, and actions. To investigate this, we induced liver injury through administration of the hepatotoxin thioacetamide (TAA) to chow fed wild type (WT) mice and lecithin:retinol acyltransferase-deficient (Lrat(-/-)) mice that are genetically unable to accumulate hepatic retinoid stores. Within 48 h of TAA-treatment, WT mice develop liver injury as evidenced by focal necrotic areas and increases in serum ALT activity and myeloperoxidase activity in hepatic parenchyma. Simultaneously, features of hepatic encephalopathy develop, as evidenced by a 25% increase in blood ammonia and a threefold reduction of blood glucose levels. This is accompanied by reduced hepatic glutathione, and increased thiobarbituric acid reactive substances, protein carbonyl and sulfhydryl groups, and increased cytochrome P450-catalyzed hydroxylation activity and flavin-containing monooxygenase activity in microsomes prepared from WT liver. Strikingly, none of these TAA-induced effects were observed for matched Lrat(-/-) mice. To confirm that TAA hepatotoxicity depends on retinoid availability, we administered, over 48 h, four oral doses of 3000 IU retinyl acetate each to the mice. This led to the development of hepatotoxicity in Lrat(-/-) mice that was similar in extent to that observed in WT mice. Our findings establish that endogenous hepatic retinoid stores can modulate the toxicity of TAA in mice.

  14. β-Carotene and its cleavage enzyme β-carotene-15,15′-oxygenase (CMOI) affect retinoid metabolism in developing tissues

    Science.gov (United States)

    Kim, Youn-Kyung; Wassef, Lesley; Chung, Stacey; Jiang, Hongfeng; Wyss, Adrian; Blaner, William S.; Quadro, Loredana

    2011-01-01

    The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. β-Carotene is the major human dietary provitamin A. β-Carotene-15,15′-oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from β-carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that β-carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol-binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP−/− mice. Since β-carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its β-carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that β-carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.—Kim, Y.-K., Wassef, L., Chung, S., Jiang, H., Wyss, A., Blaner, W. S., Quadro, L. β-Carotene and its cleavage enzyme β-carotene-15,15′-oxygenase (CMOI) affect retinoid metabolism in developing tissues. PMID:21285397

  15. De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation.

    Directory of Open Access Journals (Sweden)

    Yangkai Duan

    Full Text Available Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs and fatty acid ethyl esters (FAEEs, and is currently produced through the transesterification reaction of methanol (or ethanol and triacylglycerols (TAGs. TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

  16. De novo Biosynthesis of Biodiesel by Escherichia coli in Optimized Fed-Batch Cultivation

    Science.gov (United States)

    Cai, Ke; Tan, Xiaoming; Lu, Xuefeng

    2011-01-01

    Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate. PMID:21629774

  17. Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

    Science.gov (United States)

    Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

    2009-02-01

    The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

  18. Nutraceutical inherent of Spinacia oleracea Linn. methanolic leaf extract ameliorates isoproterenol induced myocardial necrosis in male albino Wistar rats via mitigating inflammation.

    Science.gov (United States)

    Vutharadhi, Shivaranjani; Jolapuram, Umamaheswari; Kodidhela, Lakshmi Devi

    2017-01-01

    Cardiovascular diseases (CVDs) remain the principal cause of death in both developed and developing countries. The present study was intended to appraise the nutraceutical inherent of HPLC standardized Spinacia oleracea methanolic leaf extract (SoLE) in isoproterenol (ISO) induced male albino Wistar rats via activation of pro-inflammatory signaling pathway that drives myocardial necrosis. Biochemical analysis of ISO injected rats showed significant alterations in the activities of homocysteine, paraoxonase, lecithin cholesterol acyltransferase, C-reactive protein, myeloperoxidase and caspase-3 which were further confirmed by the histopathological examination. In addition, it also flaunted a significant increase in pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 in ISO administered rats when compared with normal control rats. Pretreatment with SoLE (100, 200, and 300mg/kg bw) along with positive control gallic acid, significantly prevented all the adverse effects in ISO administered rats in a dose dependent manner. These results also reiterated the expected amelioration of myocardial necrosis in ISO induced MI rats conveying anti-atherogenic, anti-apoptotic and anti-inflammatory activities of SoLE. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  19. Asteroid hyalosis--current state of knowledge.

    Science.gov (United States)

    Jabłońska, Anna; Ciszewska, Joanna; Kęcik, Dariusz

    2014-01-01

    The search query into the Cochrane Library, Medline, Web of Science, Embase, Scopus and ScienceDirect enabled selection of research papers addressing the issue of asteroid hyalosis published in English between 1963 and January 2014. Asteroid hyalosis is a degenerative condition of the vitreous in which small, creamy or white, spherical particles (asteroid bodies) are randomly diffused within the vitreous. They consist mainly of calcium and phosphorus and have a structure of hydroxy lapatite. In 80.2-92.0% of cases the condition affects one eye only and it occurs in 0.36-1.96% of population, mostly in patients over 50 years of age and in males. Hypercholesterolemia and hypertension are systemic risk factors, but asteroid hyalosis is postulated to occur more often in retinitis pigmentosa and Leber amaurosis caused by mutations in lecithin retinol acyltransferase gene. Asteroid hyalosis also causes calcification of some intraocular lenses--mostly silicone ones. Vitreous of patients with asteroid hyalosis shows reduced gel liquefaction and anomalous vitreoretinal adhesion.

  20. Docosahexaenoic acid is a substrate for ACAT1 and inhibits cholesteryl ester formation from oleic acid in MCF-10A cells.

    Science.gov (United States)

    Antalis, Caryl J; Arnold, Tyler; Lee, Bonggi; Buhman, Kimberley K; Siddiqui, Rafat A

    2009-01-01

    MCF-10A breast epithelial cells treated with docosahexaenoic acid (DHA) or oleic acid (OA) accumulated cytoplasmic lipid droplets containing both triacylglycerol and cholesteryl esters (CE). Interestingly, total CE mass was reduced in cells treated with DHA compared to cells treated with OA, and the CEs were rich in n-3 fatty acids. Thus, we hypothesized that DHA may be, in addition to a substrate, an inhibitor of cholesterol esterification in MCF-10A cells. We determined that the primary isoform of acyl-CoA: cholesterol acyltransferase expressed in MCF-10A cells is ACAT1. We investigated CE formation with DHA, OA, and the combination in intact cells and isolated microsomes. In both cells and microsomes, the rate of CE formation was faster and more CE was formed with OA compared to DHA. DHA substantially reduced CE formation when given in combination with OA. These data suggest for the first time that DHA can act as a substrate for ACAT1. In the manner of a poor substrate, DHA also inhibited the activity of ACAT1, a universally expressed enzyme involved in intracellular cholesterol homeostasis, in a cell type that does not secrete lipids or express ACAT2.

  1. Tubulointerstitial nephritis is a dominant feature of hereditary apolipoprotein A-I amyloidosis.

    Science.gov (United States)

    Gregorini, Gina; Izzi, Claudia; Ravani, Pietro; Obici, Laura; Dallera, Nadia; Del Barba, Andrea; Negrinelli, Alessandro; Tardanico, Regina; Nardi, Matilde; Biasi, Luciano; Scalvini, Tiziano; Merlini, Giampaolo; Scolari, Francesco

    2015-06-01

    Apolipoprotein A-I is the main protein of high-density lipoprotein particles, and is encoded by the APOA1 gene. Several APOA1 mutations have been found, either affecting the lecithin:cholesterol acyltransferase activity, determining familial HDL deficiency, or resulting in amyloid formation with prevalent deposits in the kidney and liver. Evaluation of familial tubulointerstitial nephritis in patients with the Leu75Pro APOA-I amyloidosis mutation resulted in the identification of 253 carriers belonging to 50 families from Brescia, Italy. A total of 219 mutation carriers underwent clinical, laboratory, and instrumental tests. Of these, 62% had renal, hepatic, and testicular disease; 38% were asymptomatic. The disease showed an age-dependent penetrance. Tubulointerstitial nephritis was diagnosed in 49% of the carriers, 13% of whom progressed to kidney failure requiring dialysis. Hepatic involvement with elevation of cholestasis indices was diagnosed in 30% of the carriers, 38% of whom developed portal hypertension. Impaired spermatogenesis and hypogonadism was found in 68% of male carriers. The cholesterol levels were lower than normal in 80% of the mutation carriers. Thus, tubulointerstitial nephritis was highly prevalent in this large series of patients with Leu75Pro apoA-I amyloidosis. Persistent elevation of alkaline phosphatase, reduced HDL cholesterol plasma levels, and hypogonadism in men are key diagnostic features of this form of amyloidosis.

  2. In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

    Science.gov (United States)

    Deshmukh, Amit T; Verheijen, Peter J T; Maleki Seifar, Reza; Heijnen, Joseph J; van Gulik, Walter M

    2015-11-01

    In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT). Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  3. ACTTS3 encoding a polyketide synthase is essential for the biosynthesis of ACT-toxin and pathogenicity in the tangerine pathotype of Alternaria alternata.

    Science.gov (United States)

    Miyamoto, Y; Masunaka, A; Tsuge, T; Yamamoto, M; Ohtani, K; Fukumoto, T; Gomi, K; Peever, T L; Tada, Y; Ichimura, K; Akimitsu, K

    2010-04-01

    The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerine and tangerine hybrids. Sequence analysis of a genomic BAC clone identified part of the ACT-toxin TOX (ACTT) gene cluster, and knockout experiments have implicated several open reading frames (ORF) contained within the cluster in the biosynthesis of ACT-toxin. One of the ORF, designated ACTTS3, encoding a putative polyketide synthase, was isolated by rapid amplification of cDNA ends and genomic/reverse transcription-polymerase chain reactions using the specific primers designed from the BAC sequences. The 7,374-bp ORF encodes a polyketide synthase with putative beta-ketoacyl synthase, acyltransferase, methyltransferase, beta-ketoacyl reductase, and phosphopantetheine attachment site domains. Genomic Southern blots demonstrated that ACTTS3 is present on the smallest chromosome in the tangerine pathotype of A. alternata, and the presence of ACTTS3 is highly correlated with ACT-toxin production and pathogenicity. Targeted gene disruption of two copies of ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS3 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

  4. Polyketide synthases of bacterial symbionts in sponges--evolution-based applications in natural products research.

    Science.gov (United States)

    Hochmuth, Thomas; Piel, Jörn

    2009-01-01

    Marine sponges are an unusually rich source of bioactive natural products with clinical potential. They also often harbor rich communities of symbiotic bacteria that have often been suspected as the true producers of sponge-derived compounds. To date, these bacteria can in most cases not be cultivated, but culture-independent methods, such as isolating and analyzing biosynthetic gene clusters using metagenomic strategies, have recently provided first insights into their chemical potential. This review summarizes recent work of our laboratory on the study of polyketide synthases (PKSs). These studies revealed two evolutionarily distinct, unusual PKS types that are commonly found in sponge metagenomes and were shown to be of bacterial origin. One, the sup PKS, dominates sponge metagenomic DNA libraries, occurs widespread in bacteriosponges and is to date exclusively known from such animals. Data suggest that it is a type of synthase that generates methyl-branched fatty acids, which are commonly present in sponges. The other PKS type, termed trans-acyltransferase (AT) PKS, is responsible for the biosynthesis of complex, bioactive polyketides, such as the onnamides, and also occurs in free-living bacteria. The diversity of PKS genes present in a single sponge metagenome can be enormous. However, the phylogenetic approaches outlined in this review can provide valuable insights into the PKS function and structures of polyketides and can assist in the targeted isolation of gene clusters.

  5. Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study

    Directory of Open Access Journals (Sweden)

    Su-Myat Khine K

    2010-06-01

    Full Text Available Abstract Background Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD, Alzheimer's disease (AD, and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer. Results Using plasmalogen deficient (NRel-4 and plasmalogen sufficient (HEK293 cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA-containing ethanolamine plasmalogen (PlsEtn present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1 levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA reductase inhibition. Conclusion The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

  6. De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation.

    Science.gov (United States)

    Duan, Yangkai; Zhu, Zhi; Cai, Ke; Tan, Xiaoming; Lu, Xuefeng

    2011-01-01

    Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

  7. Expression of Umbelopsis ramanniana DGAT2A in seed increases oil in soybean.

    Science.gov (United States)

    Lardizabal, Kathryn; Effertz, Roger; Levering, Charlene; Mai, Jennifer; Pedroso, M C; Jury, Tom; Aasen, Eric; Gruys, Ken; Bennett, Kristen

    2008-09-01

    Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

  8. 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...... and explain how cells switch neutral lipid metabolism from storage to consumption.......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...

  9. 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.

  10. Antilipotoxicity Activity of Osmanthus fragrans and Chrysanthemum morifolium Flower Extracts in Hepatocytes and Renal Glomerular Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Po-Jung Tsai

    2017-01-01

    Full Text Available The excess influx of free fatty acids (FFAs into nonadipose tissues, such as those of liver and kidney, induces lipotoxicity leading to hepatic steatosis and renal dysfunction. The aim of this study was to investigate the protective effects of methanolic flower extracts of Osmanthus fragrans (OF and Chrysanthemum morifolium (CM against FFA-induced lipotoxicity in hepatocytes (human HepG2 cells and renal glomerular mesangial cells (mouse SV40-Mes13 cells. The results showed that OF and CM significantly suppressed FFA-induced intracellular triacylglycerol accumulation via partially inhibiting the gene expression of sterol regulatory element-binding protein-1c (SREBP-1c and glycerol-3-phosphate acyltransferase (GPAT in HepG2 cells. Both extracts inhibited reactive oxygen species (ROS generation by FFA-stimulated HepG2 cells. OF and CM also suppressed the mRNA expression of interleukin- (IL- 1β, IL-6, IL-8, tumor necrosis factor- (TNF- α, and transforming growth factor- (TGF- β by HepG2 cells treated with conditioned medium derived from lipopolysaccharide-treated THP-1 monocytes. Furthermore, OF and CM effectively inhibited oleate-induced cellular lipid accumulation, TGF-β secretion, and overexpression of fibronectin in mesangial cells. In conclusion, OF and CM possess hepatoprotective activity by inhibiting hepatic fat load and inflammation and renal protection by preventing FFA-induced mesangial extracellular matrix formation.

  11. Proteomics Analysis Reveals an Important Role for the PPAR Signaling Pathway in DBDCT-Induced Hepatotoxicity Mechanisms

    Directory of Open Access Journals (Sweden)

    Yunlan Li

    2017-07-01

    Full Text Available A patented organotin di-n-butyl-di-(4-chlorobenzohydroxamatotin (DBDCT with high a antitumor activity was designed, however, its antitumor and toxic mechanisms have not yet been clearly illustrated. Hepatic proteins of DBDCT-treated rats were identified and analyzed using LC–MS/MS with label-free quantitative technology. In total, 149 differentially expressed proteins were successfully identified. Five protein and mRNA expressions were involved in the peroxisome proliferator-activated receptor (PPAR signaling pathway, including a scavenger receptor (CD36, adipocyte fatty acid binding protein 4 (FABP4, enoyl-CoA hydratase (EHHADH, acetyl-CoA acyltransferase 1 (ACAA1, and phosphoenolpyruvate carboxykinase (PEPCK in DBDCT-treated Rat Liver (BRL cells. PPAR-α and PPAR-λ were also significantly decreased at both protein and mRNA levels. Furthermore, compared with the DBDCT treatment group, a special blocking agent of PPAR-λ T0070907 was used to evaluate the relationship between PPAR-λ and its downstream genes. Our studies indicated that DBDCT may serve as a modulator of PPAR-λ, further up-regulating CD36, FABP4 and EHHADH on the PPAR signal pathway.

  12. Adipose differentiation-related protein regulates lipids and insulin in pancreatic islets.

    Science.gov (United States)

    Faleck, D M; Ali, K; Roat, R; Graham, M J; Crooke, R M; Battisti, R; Garcia, E; Ahima, R S; Imai, Y

    2010-08-01

    The excess accumulation of lipids in islets is thought to contribute to the development of diabetes in obesity by impairing beta-cell function. However, lipids also serve a nutrient function in islets, and fatty acids acutely increase insulin secretion. A better understanding of lipid metabolism in islets will shed light on complex effects of lipids on beta-cells. Adipose differentiation-related protein (ADFP) is localized on the surface of lipid droplets in a wide range of cells and plays an important role in intracellular lipid metabolism. We found that ADFP was highly expressed in murine beta-cells. Moreover, islet ADFP was increased in mice on a high-fat diet (3.5-fold of control) and after fasting (2.5-fold of control), revealing dynamic changes in ADFP in response to metabolic cues. ADFP expression was also increased by addition of fatty acids in human islets. The downregulation of ADFP in MIN6 cells by antisense oligonucleotide (ASO) suppressed the accumulation of triglycerides upon fatty acid loading (56% of control) along with a reduction in the mRNA levels of lipogenic genes such as diacylglycerol O-acyltransferase-2 and fatty acid synthase. Fatty acid uptake, oxidation, and lipolysis were also reduced by downregulation of ADFP. Moreover, the reduction of ADFP impaired the ability of palmitate to increase insulin secretion. These findings demonstrate that ADFP is important in regulation of lipid metabolism and insulin secretion in beta-cells.

  13. Acute Testosterone Deficiency Alters Adipose Tissue Fatty Acid Storage.

    Science.gov (United States)

    Santosa, Sylvia; Bush, Nikki C; Jensen, Michael D

    2017-08-01

    Although the long-term effects of testosterone on adipose tissue lipid metabolism in men have been defined, the short-term regulation of these effects is not well understood. We examined the effects of acute testosterone withdrawal on subcutaneous abdominal and femoral adipose tissue fatty acid (FA) storage and cellular mechanisms. This was a prospective, randomized trial. Mayo Clinic Clinical Research Unit. Thirty-two male volunteers ages 18 to 50 participated in these studies. Volunteers were randomized to receive (1) no treatment (control), (2) injections (7.5 mg) of Lupron®, or (3) Lupron and testosterone (L+T) replacement for 49 days, resulting in 4 weeks of sex steroid suppression in the Lupron group. We measured body composition, fat cell size, adipose tissue meal FA and direct free FA storage, lipoprotein lipase (LPL), acyl coenzyme A synthetase (ACS), diacylglycerol acyltransferase activities, and CD36 content. Compared with control and L+T groups, acute testosterone deficiency resulted in greater femoral adipose tissue meal FA storage rates, fasting and fed LPL activity, and ACS activity. These results suggest that in men, testosterone plays a tonic role in restraining FA storage in femoral adipose tissue via suppression of LPL and ACS activities. FA storage mechanisms in men appear sensitive to short-term changes in testosterone concentrations.

  14. Does exposure to testosterone significantly alter endogenous metabolism in the marine mussel Mytilus galloprovincialis?

    Science.gov (United States)

    Fernandes, Denise; Navarro, Juan Carlos; Riva, Consuelo; Bordonali, Silvia; Porte, Cinta

    2010-11-15

    Mussels (Mytilus galloprovincialis) were exposed to different concentrations of testosterone (T: 20, 200 and 2000ng/L) in a semi-static water regime (1-day dosing intervals) for up to 5 days in an attempt to see whether endogenous steroid levels and steroid metabolism were altered by exogenous exposure to testosterone. Whole tissue levels of total testosterone (free+esterified) sharply increased in a concentration-dependent manner, from 2ng/g in controls to 290ng/g in organisms exposed to the highest concentration. In contrast, levels of free testosterone were only significantly elevated at the high-exposure group (5-fold increase with respect to controls). Increased activity of palmitoyl-CoA:testosterone acyltransferase (ATAT) was detected in organisms exposed to the highest concentration of testosterone, while those exposed to low and medium concentrations showed significant alterations in their polyunsaturated fatty acid profiles. The obtained results suggest that esterification of the excess of T with fatty acids might act as a homeostatic mechanism to maintain endogenous levels of free T stable. Interestingly, a decrease in CYP3A-like activity was detected in T-exposed mussels together with a significant decrease in the metabolism of the androgen precursor androstenedione to dihydrotestosterone (5α-DHT). Overall, the work contributes to the better knowledge of androgen metabolism in mussels. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Preclinical pharmacokinetic characterization of 2-(4-(4-(5-(2-phenyl-5-(trifluoromethyl)oxazole-4-carboxamido)-1H-benzo[d]imidazol-2-yl)phenyl)cyclohexyl) acetic acid, a novel DGAT-1 inhibitor.

    Science.gov (United States)

    Kwak, Eun-Young; Im, So Hee; Seo, Hyewon; Cho, Woon-Ki; Lee, Ye-Lim; Woo, Jaechun; Ahn, Sunjoo; Ahn, Sung-Hoon; Kwak, Hyun Jung; Ahn, Jin Hee; Bae, Myung Ae; Song, Jin Sook

    2014-05-01

    1. A novel diacylglyceride acyltransferase-1 (DGAT-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), was synthesized for a potential therapeutic use against several metabolic disorders, such as obesity, insulin resistance, and type II diabetes, characterized by excessive triglycerides (TGs) in the blood. 2. The half-lives against phase I metabolism were measured as 75.3 ± 20.9 min and over 120 min in rat and human liver microsomes, respectively. In Caco-2 cell monolayers, extremely low permeability (99.8%). 3. With the intravenous administration of KR-69232 in rats (1, 2, and 5 mg/kg), non-linear kinetics were observed at the highest dose, with significantly higher systemic clearance, higher volume of distribution, and lower dose-normalized AUC. Following oral administration, it exhibited low bioavailability (<10%) and was absorbed slowly (T(max), 3.8-5.2 h) over the dose range. We also confirmed that considerable KR-69232 remained in the intestine at T(max), demonstrating its limited absorption into the systemic circulation.

  16. 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.

  17. Enhanced lipid accumulation and biodiesel production by oleaginous Chlorella protothecoides under a structured heterotrophic-iron (II) induction strategy.

    Science.gov (United States)

    Li, Yuqin; Mu, Jinxiu; Chen, Di; Xu, Hua; Han, Fangxin

    2015-05-01

    A structured heterotrophic-iron (II) induction (HII) strategy was proposed to enhance lipid accumulation in oleaginous Chlorella protothecoides. C. protothecoides subjected to heterotrophic-iron (II) induction achieved a favorable lipid accumulation up to 62 % and a maximum lipid productivity of 820.17 mg/day, representing 2.78-fold and 3.64-fold increase respectively over heterotrophic cultivation alone. HII-induced cells produced significantly elevated levels of 16:0, 18:1(Δ9), and 18:2(Δ9,12) fatty acids (over 90 %). The lipid contents and plant lipid-like fatty acid compositions exhibit the potential of HII-induced C. protothecoides as biodiesel feedstock. Furthermore, 31 altered proteins in HII-induced algal cells were successfully identified. These differentially expressed proteins were assigned into nine molecular function categories, including carbohydrate metabolism, lipid biosynthesis, Calvin cycle, cellular respiration, photosynthesis, energy and transport, protein biosynthesis, regulate and defense, and unclassified. Analysis using the Kyoto encyclopedia of genes and genomes and gene ontology annotation showed that malic enzyme, acyltransferase, and ACP were key metabolic checkpoints found to modulate lipid accumulation in C. protothecoides. The results provided possible applications of HII cultivation strategy in other microalgal species and new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity.

  18. The role of desaturases in the biosynthesis of marking pheromones in bumblebee males.

    Science.gov (United States)

    Buček, Aleš; Vogel, Heiko; Matoušková, Petra; Prchalová, Darina; Záček, Petr; Vrkoslav, Vladimír; Šebesta, Petr; Svatoš, Aleš; Jahn, Ullrich; Valterová, Irena; Pichová, Iva

    2013-08-01

    Bumblebee males (Hymenoptera) produce species-specific labial gland secretions called marking pheromones (MPs). MPs generally consist of terpenoids and fatty-acid-derived aliphatic compounds with various chain lengths predominantly containing one or no double bonds. The unsaturated fatty-acid-derived MP components were hypothesized to be produced by fatty acid desaturases (FADs) that exhibit diverse substrate specificities. To address this hypothesis, we isolated and functionally characterized FADs from three bumblebee species: Bombus lucorum, Bombus terrestris, and Bombus lapidarius. By employing RNA sequencing of the male labial glands and fat bodies of B. lucorum and B. terrestris, we identified five paralogous FAD-like sequences but only two FAD lineages were abundant and differentially expressed in the labial glands. We found that abundant FAD lineages were also expressed in the labial gland and fat body of Bombus lapidarius. Functional characterization of FADs in a yeast expression system confirmed that Δ4-FADs exhibited a unique Δ4-desaturase activity exclusively on 14-carbon fatty acyls and Δ9-FADs displayed Δ9-desaturase activity on 14- to 18-carbon fatty acyls. These results indicate that Δ9-FADs are involved in the biosynthesis of major unsaturated components of MPs in B. lucorum and B. lapidarius despite the diverse MP composition of these bumblebee species. The contribution of lipases, acyltransferases, esterases, and fatty acid reductases to production of the species-specific MP composition is also discussed in light of the transcriptomic data obtained in this study. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth

    Science.gov (United States)

    Trentacoste, Emily M.; Shrestha, Roshan P.; Smith, Sarah R.; Glé, Corine; Hartmann, Aaron C.; Hildebrand, Mark; Gerwick, William H.

    2013-01-01

    Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type–like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth. PMID:24248374

  20. Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells.

    Science.gov (United States)

    Cho, Young Sik; Kim, Chi Hyun; Kim, Ki Young; Cheon, Hyae Gyeong

    2012-05-01

    Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.