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Sample records for alcohol acyltransferase awat

  1. Suppression of PPARγ-mediated monoacylglycerol O-acyltransferase 1 expression ameliorates alcoholic hepatic steatosis

    PubMed Central

    Yu, Jung Hwan; Song, Su Jin; Kim, Ara; Choi, Yoonjeong; Seok, Jo Woon; Kim, Hyo Jung; Lee, Yoo Jeong; Lee, Kwan Sik; Kim, Jae-woo

    2016-01-01

    Alcohol consumption is one of the major causes of hepatic steatosis, fibrosis, cirrhosis, and superimposed hepatocellular carcinoma. Ethanol metabolism alters the NAD+/NADH ratio, thereby suppressing the activity of sirtuin family proteins, which may affect lipid metabolism in liver cells. However, it is not clear how long-term ingestion of ethanol eventually causes lipid accumulation in liver. Here, we demonstrate that chronic ethanol ingestion activates peroxisome proliferator-activated receptor γ (PPARγ) and its target gene, monoacylglycerol O-acyltransferase 1 (MGAT1). During ethanol metabolism, a low NAD+/NADH ratio repressed NAD-dependent deacetylase sirtuin 1 (SIRT1) activity, concomitantly resulting in increased acetylated PPARγ with high transcriptional activity. Accordingly, SIRT1 transgenic mice exhibited a low level of acetylated PPARγ and were protected from hepatic steatosis driven by alcohol or PPARγ2 overexpression, suggesting that ethanol metabolism causes lipid accumulation through activation of PPARγ through acetylation. Among the genes induced by PPARγ upon alcohol consumption, MGAT1 has been shown to be involved in triglyceride synthesis. Thus, we tested the effect of MGAT1 knockdown in mice following ethanol consumption, and found a significant reduction in alcohol-induced hepatic lipid accumulation. These results suggest that MGAT1 may afford a promising approach to the treatment of fatty liver disease. PMID:27404390

  2. Suppression of PPARγ-mediated monoacylglycerol O-acyltransferase 1 expression ameliorates alcoholic hepatic steatosis.

    PubMed

    Yu, Jung Hwan; Song, Su Jin; Kim, Ara; Choi, Yoonjeong; Seok, Jo Woon; Kim, Hyo Jung; Lee, Yoo Jeong; Lee, Kwan Sik; Kim, Jae-Woo

    2016-01-01

    Alcohol consumption is one of the major causes of hepatic steatosis, fibrosis, cirrhosis, and superimposed hepatocellular carcinoma. Ethanol metabolism alters the NAD(+)/NADH ratio, thereby suppressing the activity of sirtuin family proteins, which may affect lipid metabolism in liver cells. However, it is not clear how long-term ingestion of ethanol eventually causes lipid accumulation in liver. Here, we demonstrate that chronic ethanol ingestion activates peroxisome proliferator-activated receptor γ (PPARγ) and its target gene, monoacylglycerol O-acyltransferase 1 (MGAT1). During ethanol metabolism, a low NAD(+)/NADH ratio repressed NAD-dependent deacetylase sirtuin 1 (SIRT1) activity, concomitantly resulting in increased acetylated PPARγ with high transcriptional activity. Accordingly, SIRT1 transgenic mice exhibited a low level of acetylated PPARγ and were protected from hepatic steatosis driven by alcohol or PPARγ2 overexpression, suggesting that ethanol metabolism causes lipid accumulation through activation of PPARγ through acetylation. Among the genes induced by PPARγ upon alcohol consumption, MGAT1 has been shown to be involved in triglyceride synthesis. Thus, we tested the effect of MGAT1 knockdown in mice following ethanol consumption, and found a significant reduction in alcohol-induced hepatic lipid accumulation. These results suggest that MGAT1 may afford a promising approach to the treatment of fatty liver disease. PMID:27404390

  3. Computational study enlightens the structural role of the alcohol acyltransferase DFGWG motif.

    PubMed

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

    2015-08-01

    Alcohol acyltransferases (AAT) catalyze the esterification reaction of alcohols and acyl-CoA into esters in fruits and flowers. Despite the high divergence between AAT enzymes, two important and conserved motifs are shared: the catalytic HxxxD motif, and the DFGWG motif. The latter is proposed to play a structural role; however, its function remains unclear. The DFGWG motif is located in loop 21 and stabilized by a hydrogen bond between residues Y52 and D381. Also, this motif is distant from the HxxxD motif, and most probably without a direct role in the substrate interaction. To evaluate the role of the DFGWG motif, in silico analysis was performed in the VpAAT1 protein. Three mutants (Y52F, D381A and D381E) were evaluated. Major changes (size and shape) in the solvent channels were found, although no differences were revealed in the entire 3D structure. Molecular dynamics simulations and docking studies described unfavorable energies for interaction of the mutant proteins with different substrates, as well as unfavored ligand orientations in the solvent channel. Additionally, we examined the contribution of different energetic parameters to the total free energy of protein-ligand complexes by the MM-GBSA method. The complexes differed mainly in their van der Waals contributions and have unfavorable electrostatic interactions. VpAAT1, Y52F and D381A mutants showed a dramatic reduction in the binding capacity to several substrates, which is related to differences in electrostatic potential on the protein surfaces, suggesting that D381 from the DFGWG motif and residue Y52 play a crucial role in maintenance of the adequate solvent channel structure required for catalysis. Graphical abstract Molecular docking, molecular dynamics (MD) simulations and MM-GBSA free energy calculations were employed to obtain quantitative estimates for the binding free energies of wild type Vasconcellea pubescens alcohol acyltransferase (VpAAT1-WT) and the protein mutants. Left VpAAT1

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

    PubMed

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

    2016-05-01

    Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels. PMID:26814045

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Herrera, Raúl; Caballero, Julio; Alzate-Morales, Jans H.

    2016-01-01

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

  7. Divergence in the Enzymatic Activities of a Tomato and Solanum pennellii Alcohol Acyltransferase Impacts Fruit Volatile Ester Composition.

    PubMed

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

    2014-10-29

    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species S. pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrates preferences that explain variations observed in the volatiles. Together, the results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of synthesis and degradation of esters. PMID:25355057

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

    PubMed

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

    2015-01-01

    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species Solanum pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrate preferences that explain the variations observed in the volatiles. The results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of the synthesis and degradation of esters. PMID:25578279

  9. Bioengineering recombinant diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 115 DGAT sequences are identified from 69 organisms in the GenBank databases. Only a few papers have been published in the last 28 years on the exp...

  10. Acyltransferases in bacteria.

    PubMed

    Röttig, Annika; Steinbüchel, Alexander

    2013-06-01

    Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue. PMID:23699259

  11. Acyltransferases in Bacteria

    PubMed Central

    Röttig, Annika

    2013-01-01

    SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue. PMID:23699259

  12. Technical note: Improving the AWAT filter with interpolation schemes for advanced processing of high resolution data

    NASA Astrophysics Data System (ADS)

    Peters, Andre; Nehls, Thomas; Wessolek, Gerd

    2016-06-01

    Weighing lysimeters with appropriate data filtering yield the most precise and unbiased information for precipitation (P) and evapotranspiration (ET). A recently introduced filter scheme for such data is the AWAT (Adaptive Window and Adaptive Threshold) filter (Peters et al., 2014). The filter applies an adaptive threshold to separate significant from insignificant mass changes, guaranteeing that P and ET are not overestimated, and uses a step interpolation between the significant mass changes. In this contribution we show that the step interpolation scheme, which reflects the resolution of the measuring system, can lead to unrealistic prediction of P and ET, especially if they are required in high temporal resolution. We introduce linear and spline interpolation schemes to overcome these problems. To guarantee that medium to strong precipitation events abruptly following low or zero fluxes are not smoothed in an unfavourable way, a simple heuristic selection criterion is used, which attributes such precipitations to the step interpolation. The three interpolation schemes (step, linear and spline) are tested and compared using a data set from a grass-reference lysimeter with 1 min resolution, ranging from 1 January to 5 August 2014. The selected output resolutions for P and ET prediction are 1 day, 1 h and 10 min. As expected, the step scheme yielded reasonable flux rates only for a resolution of 1 day, whereas the other two schemes are well able to yield reasonable results for any resolution. The spline scheme returned slightly better results than the linear scheme concerning the differences between filtered values and raw data. Moreover, this scheme allows continuous differentiability of filtered data so that any output resolution for the fluxes is sound. Since computational burden is not problematic for any of the interpolation schemes, we suggest always using the spline scheme.

  13. Characterization of cultivar differences in alcohol acyltransferase and 1-aminocyclopropane-1carboxylate synthase gene expression and volatile compound emission during apple fruit maturation and ripening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alcohol acyl transferase (AAT) catalyzes the last step of volatile ester biosynthesis, and ethylene purportedly regulates AAT gene expression. In this study, expession patterns of four apple AAT genes and two ethylene biosynthesis genes were investigated in two apple cultivars with relatively high ...

  14. Sequence analysis of diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the final step of triacylglycerol (TAG) biosynthesis in eukaryotes. DGATs esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA. Plants and animals deficient in DGATs accumulate less TAG and over-expression of DGATs increases TAG. DGAT knock...

  15. Microscopic and thermodynamic properties of the HFA134a-water interface: atomistic computer simulations and tensiometry under pressure.

    PubMed

    Peguin, Robson P S; Selvam, Parthiban; da Rocha, Sandro R P

    2006-10-10

    A combined computational and experimental approach is used to determine the interfacial thermodynamic and structural properties of the liquid 1,1,1,2-tetrafluoroethane (HFA134a)-vapor and liquid HFA134a-water (HFA134a|W) interfaces at 298 K and saturation pressure. Molecular dynamics (MD) computer simulations reveal a stable interface between HFA134a and water. The "10-90" interfacial thickness is comparable with those typically reported for organic-water systems. The interfacial tension of the HFA134a|W interface obtained from the pressure tensor analysis of the MD trajectory is in good agreement with the experimental value determined using in situ high-pressure tensiometry. These results indicate that the potential models utilized are capable of describing the intermolecular interactions between these two fluids. The tension of the HFA134a|W interface is significantly lower than those typically observed for conventional oil-water interfaces and similar to that of the compressed CO(2)-water interface, observed at moderate CO(2) pressures. The MD and tensiometric results are also compared and contrasted with the HFA134a|W and chlorofluorocarbon-water tension values estimated from a parametric relationship. This represents the first report of the interfacial and microscopic properties of the (propellant) hydrofluoroalkanes (HFA)|W interface. The results presented here are of relevance in the design of surfactants capable of forming and stabilizing water-in-HFA microemulsions. Reverse aqueous microemulsions in HFA-based pressurized metered-dose inhalers are candidate formulations for the systemic delivery of biomolecules to and through the lungs. PMID:17014124

  16. Membrane topology of hedgehog acyltransferase.

    PubMed

    Matevossian, Armine; Resh, Marilyn D

    2015-01-23

    Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors. PMID:25488661

  17. Serine carboxypeptidase-like acyltransferases from plants.

    PubMed

    Mugford, Sam T; Milkowski, Carsten

    2012-01-01

    Serine carboxypeptidase-like (SCPL) acyltransferases facilitate transacylation reactions using energy-rich 1-O-β-glucose esters in the synthesis of an array of bioactive compounds and are associated with the diversification of plant natural products. SCPL acyltransferases have evolved from a hydrolytic ancestor by adapting functional elements of the proteases such as the catalytic triad, oxyanion hole, and substrate recognition H-bond network to their new function. As vacuolar proteins, SCPL acyltransferases define an alternative cellular route of transacylation spatially separated from the cytoplasmic enzymes of the BAHD acyltransferase family named according to the first characterized members (BEAT, AHCT, HCBT, and DAT). Recent efforts in cloning and characterization led to the identification of diagnostic peptides for SCPL acyltransferases, enabling the detection of candidate genes in several plant genomes. Detailed biochemical analysis of SCPL acyltransferases is strongly dependent on comprehensive heterologous expression systems, efficient protein purification protocols, and the supply of appropriate substrates. This chapter describes some useful techniques and strategies for identification and characterization of SCPL acyltransferases. PMID:23034234

  18. Alcohol

    MedlinePlus

    ... How Can I Help a Friend Who Cuts? Alcohol KidsHealth > For Teens > Alcohol Print A A A ... you can make an educated choice. What Is Alcohol? Alcohol is created when grains, fruits, or vegetables ...

  19. Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase

    PubMed Central

    Kawelke, Steffen; Feussner, Ivo

    2015-01-01

    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. PMID:26714272

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

    PubMed Central

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

    2005-01-01

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

  1. Alcohol

    MedlinePlus

    ... Text Size: A A A Listen En Español Alcohol Wondering if alcohol is off limits with diabetes? Most people with diabetes can have a moderate amount of alcohol. Research has shown that there can be some ...

  2. Alcohol

    MedlinePlus

    If you are like many Americans, you drink alcohol at least occasionally. For many people, moderate drinking ... risky. Heavy drinking can lead to alcoholism and alcohol abuse, as well as injuries, liver disease, heart ...

  3. Alcohol

    MedlinePlus

    ... Got Homework? Here's Help White House Lunch Recipes Alcohol KidsHealth > For Kids > Alcohol Print A A A Text Size What's in ... What Is Alcoholism? Say No en español El alcohol Getting the Right Message "Hey, who wants a ...

  4. Alcohol

    MedlinePlus

    ... as well as injuries, liver disease, heart disease, cancer, and other health problems. It can also cause problems at home, at work, and with friends. NIH: National Institute on Alcohol Abuse and Alcoholism

  5. Alcoholism.

    ERIC Educational Resources Information Center

    Caliguri, Joseph P., Ed.

    This extensive annotated bibliography provides a compilation of documents retreived from a computerized search of the ERIC, Social Science Citation Index, and Med-Line databases on the topic of alcoholism. The materials address the following areas of concern: (1) attitudes toward alcohol users and abusers; (2) characteristics of alcoholics and…

  6. Synthesis of Novel Acylglycerol Substrates for Acyltransferases.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    1,2-Diacylglycerols (DG) are the native substrates for the diacylglycerol acyltransferase (DGAT). It is difficult to chemically synthesize DG containing hydroxy fatty acids in specific positions on the glycerol backbone. An alternate approach is to start from acylglycerols containing hydroxy fatty...

  7. Bioengineering recombinant tung tree diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Plants and animals defici...

  8. Expression and purification of diacylglycerol acyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG and over-expression of DGATs increases TAG. DGAT knockout mice are resistant to ...

  9. Enhancing the Acyltransferase Activity of Candida antarctica Lipase A by Rational Design.

    PubMed

    Müller, Janett; Sowa, Miriam A; Fredrich, Birte; Brundiek, Henrike; Bornscheuer, Uwe T

    2015-08-17

    A few lipases, such as Candida antarctica lipase A (CAL-A), are known to possess acyltransferase activity. This enables the enzyme to synthesize fatty acid esters from natural oils and alcohols even in the presence of bulk water. Unfortunately, fatty acids are still formed in these reactions as undesired side-products. To reduce the amount of fatty acids, several CAL-A variants were rationally designed based on its crystal structure. These variants were expressed in Escherichia coli and Pichia pastoris, purified, and their acyltransferase/hydrolase activities were investigated by various biocatalytic approaches. Among the investigated variants, mutant Asp122Leu showed a significant decrease in the hydrolytic activity, thus reducing the side-product yield during acylation. As desired, this variant retained wild-type process-relevant features like pH profile and thermostability. PMID:26058745

  10. Membrane bound O-acyltransferases and their inhibitors.

    PubMed

    Masumoto, Naoko; Lanyon-Hogg, Thomas; Rodgers, Ursula R; Konitsiotis, Antonios D; Magee, Anthony I; Tate, Edward W

    2015-04-01

    Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes. PMID:25849925

  11. Alcohol.

    ERIC Educational Resources Information Center

    Schibeci, Renato

    1996-01-01

    Describes the manufacturing of ethanol, the effects of ethanol on the body, the composition of alcoholic drinks, and some properties of ethanol. Presents some classroom experiments using ethanol. (JRH)

  12. Acyl-coenzyme A:cholesterol acyltransferases

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    Golczak, Marcin; Kiser, Philip D.; Sears, Avery E.; Lodowski, David T.; Blaner, William S.; Palczewski, Krzysztof

    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.

  14. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis

    PubMed Central

    2011-01-01

    Background Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. Results We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. Conclusions In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa

  15. Soybean oil biosynthesis: role of diacylglycerol acyltransferases.

    PubMed

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

    2013-03-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to form seed oil triacylglycerol (TAG). To understand the features of genes encoding soybean (Glycine max) DGATs and possible roles in soybean seed oil synthesis and accumulation, two full-length cDNAs encoding type 1 diacylglycerol acyltransferases (GmDGAT1A and GmDGAT1B) were cloned from developing soybean seeds. These coding sequences share identities of 94 % and 95 % in protein and DNA sequences. The genomic architectures of GmDGAT1A and GmDGAT1B both contain 15 introns and 16 exons. Differences in the lengths of the first exon and most of the introns were found between GmDGAT1A and GmDGAT1B genomic sequences. Furthermore, detailed in silico analysis revealed a third predicted DGAT1, GmDGAT1C. GmDGAT1A and GmDGAT1B were found to have similar activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-diacylglycerol were preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. Both transcripts are much more abundant in developing seeds than in other tissues including leaves, stem, roots, and flowers. Both soybean DGAT1A and DGAT1B are highly expressed at developing seed stages of maximal TAG accumulation with DGAT1B showing highest expression at somewhat later stages than DGAT1A. DGAT1A and DGAT1B show expression profiles consistent with important roles in soybean seed oil biosynthesis and accumulation. PMID:23322364

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

    PubMed

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

    2014-06-01

    Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein. PMID:24748621

  17. Thematic Review Series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis*

    PubMed Central

    Zhang, Yong-Mei; Rock, Charles O.

    2008-01-01

    Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase. PMID:18369234

  18. Lecithin retinol acyltransferase forms functional homodimers.

    PubMed

    Jahng, Wan Jin; Cheung, Eric; Rando, Robert R

    2002-05-21

    Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation. PMID:12009892

  19. Human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Jauhiainen, M.; Stevenson, K.J.; Dolphin, P.J.

    1988-05-15

    Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. The authors proposed a covalent catalytic mechanism of action for LCAT in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols they have probed the geometry of the catalytic site. They conclude that the two catalytic cysteine residues of LCAT (Cys/sup 31/ and Cys /sup 184/) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by teh bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.

  20. Evolution of serine carboxypeptidase-like acyltransferases in the monocots

    PubMed Central

    Mugford, Sam T

    2010-01-01

    The serine carboxypeptidases are a large family of proteases. in higher plants some members of this family have diversified and adopted new functions as acyltransferases required for the synthesis of natural products. we recently reported the first serine carboxypeptidase-like (scpl) acyltransferase enzyme to be characterized from monocotyledonous plants.1 This enzyme, AsSCPL1, is required for acylation of antimicrobial terpenes (avenacins) that are produced in the roots of oat (Avena spp.) and that provide protection against soil-borne pathogens. The SCPL acyltransferase enzyme family has undergone substantial expansion following the divergence of monocots and dicots. Here we discuss the evolution of this SCPL enzyme family in monocots, their contribution to metabolic diversity, and the roles of these enzymes in biotic and abiotic stress tolerance. PMID:20173416

  1. Diacylglycerol Acyltransferase-2 (DGAT2) and Monoacylglycerol Acyltransferase-2 (MGAT2) Interact to Promote Triacylglycerol Synthesis*

    PubMed Central

    Jin, Youzhi; McFie, Pamela J.; Banman, Shanna L.; Brandt, Curtis; Stone, Scot J.

    2014-01-01

    Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis. PMID:25164810

  2. Diacylglycerol acyltransferase-2 (DGAT2) and monoacylglycerol acyltransferase-2 (MGAT2) interact to promote triacylglycerol synthesis.

    PubMed

    Jin, Youzhi; McFie, Pamela J; Banman, Shanna L; Brandt, Curtis; Stone, Scot J

    2014-10-10

    Acyl CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is an integral membrane protein that catalyzes triacylglycerol (TG) synthesis using diacylglycerol and fatty acyl CoA as substrates. DGAT2 resides in the endoplasmic reticulum (ER), but when cells are incubated with fatty acids, DGAT2 interacts with lipid droplets presumably to catalyze localized TG synthesis for lipid droplet expansion. Previous studies have shown that DGAT2 interacts with proteins that synthesize its fatty acyl CoA substrates. In this study, we provide additional evidence that DGAT2 is present in a protein complex. Using a chemical cross-linker, disuccinimidyl suberate (DSS), we demonstrated that DGAT2 formed a dimer and was also part of a protein complex of ∼ 650 kDa, both in membranes and on lipid droplets. Using co-immunoprecipitation experiments and an in situ proximity ligation assay, we found that DGAT2 interacted with monoacylglycerol acyltransferase (MGAT)-2, an enzyme that catalyzes the synthesis of diacylglycerol. Deletion mutagenesis showed that the interaction with MGAT2 was dependent on the two transmembrane domains of DGAT2. No significant interaction of DGAT2 with lipin1, another enzyme that synthesizes diacylglycerol, could be detected. When co-expressed in cells, DGAT2 and MGAT2 co-localized in the ER and on lipid droplets. Co-expression also resulted in increased TG storage compared with expression of DGAT2 or MGAT2 alone. Incubating McArdle rat hepatoma RH7777 cells with 2-monoacylglycerol caused DGAT2 to translocate to lipid droplets. This also led to the formation of large cytosolic lipid droplets, characteristic of DGAT2, but not DGAT1, and indicated that DGAT2 can utilize monoacylglycerol-derived diacylglycerol. These findings suggest that the interaction of DGAT2 and MGAT2 serves to channel lipid substrates efficiently for TG biosynthesis. PMID:25164810

  3. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG. Over-expression of DGATs increases TAG. DGAT knockout mice are resistant to diet-induced obesity and lack milk secr...

  4. Expression and purification of membrane protein diacylglycerol acyltransferase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG. Over-expression of DGATs increases TAG in seeds and other tissues. DGAT knockout mice are resista...

  5. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) are responsible for the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Different forms of DGATs have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii), which contains approximately 80% high-valu...

  6. Purification of recombinant tung tree diacylglycerol acyltransferases from E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Over-expression of DGATs ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT isoforms have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii) which contains 80% high-value eleostearic acid in its seed oils. ...

  8. Lipoprotein products of lecithin: cholesterol acyltransferase and cholesteryl ester transfer.

    PubMed

    Rose, H G; Ellerbe, P

    1982-09-14

    High-density lipoprotein substrates and products of human plasma lecithin: cholesterol acyltransferase have been labelled with radioisotopic cholesteryl esters in order to facilitate identification. [3H]Cholesteryl esters were formed by endogenous HDL3/VHDL enzyme (d greater than 1.125 g/ml) following incubation with mixed vesicles of phosphatidylcholine, unesterified cholesterol and 3H-labelled unesterified cholesterol. Transfer of labelled esters to acceptor lipoproteins (VLDL+LDL, d less than 1.063 g/ml) was employed to distinguish a hypothetical transfer complex. Separation of labelled HDL3/VHDL was by gel-permeation chromatography. The results indicate that a subpopulation of labelled HDL3/VHDL cholesteryl esters (43-61% of total) were removed by VLDL/LDL during a 3 h transfer period and these derive from the smaller lipoproteins of the spectrum. HDL carrying non-transferable [3H]cholesteryl esters localize to the larger HDL3. Transfer rates were proportional to ratios of acceptor to donor lipoproteins. Net transfer of cholesteryl esters from the smaller HDL3 also occurred, but was smaller in magnitude (about 10.5% of total). Acyltransferase assays indicated that enzyme distribution is skewed to larger-sized HDL3, suggesting that the non-transferable components might be lecithin: cholesterol acyltransferase-containing parent complexes, while the smaller transfer products contain little acyltransferase. The results fit the hypothesis that a parent HDL3-lecithin: cholesterol acyltransferase complex generates a smaller-sized lipoprotein product which is active in cholesteryl ester transport. PMID:7126623

  9. Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity.

    PubMed

    Onorato, Thomas M; Haldar, Dipak

    2002-09-01

    Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed. PMID:12207885

  10. Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling.

    PubMed

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

    2013-04-01

    Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a new target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling. PMID:23416332

  11. Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs.

    PubMed

    Taylor, Martin S; Dempsey, Daniel R; Hwang, Yousang; Chen, Zan; Chu, Nam; Boeke, Jef D; Cole, Philip A

    2015-10-01

    Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base. PMID:26246082

  12. Biosynthesis of phosphatidylcholine by human lysophosphatidylcholine acyltransferase 1.

    PubMed

    Harayama, Takeshi; Shindou, Hideo; Shimizu, Takao

    2009-09-01

    Pulmonary surfactant is a complex of phospholipids and proteins lining the alveolar walls of the lung. It reduces surface tension in the alveoli, and is critical for normal respiration. Pulmonary surfactant phospholipids consist mainly of phosphatidylcholine (PC) and phosphatidylglycerol (PG). Although the phospholipid composition of pulmonary surfactant is well known, the enzyme(s) involved in its biosynthesis have remained obscure. We previously reported the cloning of murine lysophosphatidylcholine acyltransferase 1 (mLPCAT1) as a potential biosynthetic enzyme of pulmonary surfactant phospholipids. mLPCAT1 exhibits lysophosphatidylcholine acyltransferase (LPCAT) and lysophosphatidylglycerol acyltransferase (LPGAT) activities, generating PC and PG, respectively. However, the enzymatic activity of human LPCAT1 (hLPCAT1) remains controversial. We report here that hLPCAT1 possesses LPCAT and LPGAT activities. The activity of hLPCAT1 was inhibited by N-ethylmaleimide, indicating the importance of some cysteine residue(s) for the catalysis. We found a conserved cysteine (Cys(211)) in hLPCAT1 that is crucial for its activity. Evolutionary analyses of the close homologs of LPCAT1 suggest that it appeared before the evolution of teleosts and indicate that LPCAT1 may have evolved along with the lung to facilitate respiration. hLPCAT1 mRNA is highly expressed in the human lung. We propose that hLPCAT1 is the biosynthetic enzyme of pulmonary surfactant phospholipids. PMID:19383981

  13. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

    Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast. PMID:25265

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

    PubMed

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

    2016-02-01

    Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved. PMID:26602447

  15. Alcoholism and Alcohol Abuse

    MedlinePlus

    ... This means that their drinking causes distress and harm. It includes alcoholism and alcohol abuse. Alcoholism, or ... brain, and other organs. Drinking during pregnancy can harm your baby. Alcohol also increases the risk of ...

  16. Structure of a bacterial toxin-activating acyltransferase

    PubMed Central

    Greene, Nicholas P.; Hughes, Colin; Koronakis, Vassilis

    2015-01-01

    Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host–cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove. PMID:26016525

  17. Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein*

    PubMed Central

    Konitsiotis, Antonio D.; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W.; Magee, Anthony I.

    2015-01-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  18. Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

    PubMed

    Konitsiotis, Antonio D; Jovanović, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W; Magee, Anthony I

    2015-02-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

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

    PubMed

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

    2014-10-01

    Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation-reacylation cycle from a well-established, versatile vertebrate model species. PMID:25175316

  20. Radioassay of the stereospecificity of 2-monoacylglycerol acyltransferase

    SciTech Connect

    Manganaro, F.; Kuksis, A.; Myher, J.J.

    1982-01-01

    The 2-monoacylglycerol acyltransferase (EC 2.3.1.22, acylglycerol palmitoyl transferase) catalyzes the synthesis of X-1,2-diacylglycerols from 2-monoacylglycerol and acyl CoA with an apparently variable stereochemical specificity. A microassay for determining the ratio of sn-1,2- and sn-2,3-diacylglycerol formed by the acylation of radioactive 2-monoacylglycerol in intact cell or in cell-free systems in the presence of free fatty acids and cofactors has been developed. The diacyglycerols isolated by thin-layer chromatography using nonradioactive racemic diacylglycerols as carriers. The enantiomer content is determined following a chemical synthesis of X-1,2-diacylphosphatidylcholines and a stereospecific stepwise release of the sn-1,2- and sn-2,3-diacylglycerols by phospholipase C. By using thin-layer chromatography for the isolation of the hydrolysis products, known samples ranging in enantiomer ratios from 0.05 to 20 and containing 5000 to 200,000 cpm can be assayed to within 1% of the major and within 10% of the minor enantiomer content. The method is applicable to the determination of the enantiomer content of X-1,2-diacylglycerols generated via other acyltransferases and via lipolysis of triacylglycerols and diacylglycerolphospholipids in other biological systems.

  1. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons.

    PubMed

    Tabe, Shirou; Hikiji, Hisako; Ariyoshi, Wataru; Hashidate-Yoshida, Tomomi; Shindou, Hideo; Okinaga, Toshinori; Shimizu, Takao; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2016-07-01

    Glycerophospholipids, which are components of biomembranes, are formed de novo by the Kennedy pathway and subsequently mature through the Lands cycle. Lysophospholipid acyltransferases (LPLATs) are key enzymes in both pathways and influence the fatty acid composition of biomembranes. Neuronal differentiation is characterized by neurite outgrowth, which requires biomembrane biosynthesis. However, the role of LPLATs in neuronal differentiation remains unknown. In this study, we examined whether LPLATs are involved in neuronal differentiation using all-trans-retinoic acid (ATRA)-treated P19C6 cells. In these cells, mRNA levels of lysophosphatidylethanolamine acyltransferase (LPEAT)-1/membrane-bound O-acyltransferase (MBOAT)-1 were higher than those in undifferentiated cells. LPEAT enzymatic activity increased with 16:0- and 18:1-CoA as acyl donors. When LPEAT1/MBOAT1 was knocked down with small interfering RNA (siRNA), outgrowth of neurites and expression of neuronal markers decreased in ATRA-treated P19C6 cells. Voltage-dependent calcium channel activity was also suppressed in these cells transfected with LPEAT1/MBOAT1 siRNA. These results suggest that LPEAT1/MBOAT1 plays an important role in neurite outgrowth and function.-Tabe, S., Hikiji, H., Ariyoshi, W., Hashidate-Yoshida, T., Shindou, H., Okinaga, T., Shimizu, T., Tominaga, K., Nishihara, T. Lysophosphatidylethanolamine acyltransferase 1/membrane-bound O-acyltransferase 1 regulates morphology and function of P19C6 cell-derived neurons. PMID:27048541

  2. [Sn-glycerol-3-phosphate acyltransferases (GPATs) in plants].

    PubMed

    Liu, Cong; Xiao, Dan-Wang; Shi, Chun-Lin; Hu, Xue-Fang; Wu, Ke-Bin; Guan, Chun-Yun; Xiong, Xing-Hua

    2013-12-01

    Sn-glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation at sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LPA) in an acyl-CoA or acyl-ACP-dependent manner, which is the initial and rate-determining step of TAG biosynthetic pathway. Some GPATs have sn-2 transfer activity. Part members of the GPAT gene family have been cloned from different plant species. Based on their subcellular localizations, GPATs can be classified into three types, plastid GPATs, mitochondria GPATs and endoplasmic reticulum GPATs. GPATs exhibit diverse biochemical properties and are involved in synthesis of several lipids such as TAG, suberin, and cutin which play important roles in the growth and development of plants. This review summarized the current understanding of the chromosomal locus and gene structure of GPAT genes and the subcellular localization, sn-2 regiospecificity, substrates specialty, and functions of GPATs in plants. PMID:24645344

  3. Engineering the acyltransferase substrate specificity of assembly line polyketide synthases.

    PubMed

    Dunn, Briana J; Khosla, Chaitan

    2013-08-01

    Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active 'unnatural' natural products. PMID:23720536

  4. Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family.

    PubMed

    Körbes, Ana Paula; Kulcheski, Franceli Rodrigues; Margis, Rogério; Margis-Pinheiro, Márcia; Turchetto-Zolet, Andreia Carina

    2016-03-01

    Lysophosphatidic acid acyltransferases (LPAATs) perform an essential cellular function by controlling the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane, signaling and storage lipids. Although LPAATs have been extensively explored by functional and biotechnological studies, little is known about their molecular evolution and diversification. We performed a genome-wide analysis using data from several plants and animals, as well as other eukaryotic and prokaryotic species, to identify LPAAT genes and analyze their evolutionary history. We used phylogenetic and molecular evolution analysis to test the hypothesis of distinct origins for these genes. The reconstructed phylogeny supported the ancient origin of some isoforms (plant LPAAT1 and LPAATB; animal AGPAAT1/2), while others emerged more recently (plant LPAAT2/3/4/5; AGPAAT3/4/5/8). Additionally, the hypothesis of endosymbiotic origin of the plastidic isoform LPAAT1 was confirmed. LPAAT genes from plants and animals mainly experienced strong purifying selection pressures with limited functional divergence after the species-specific duplications. Gene expression analyses of LPAAT isoforms in model plants demonstrated distinct LPAAT expression patterns in these organisms. The results showed that distinct origins followed by diversification of the LPAAT genes shaped the evolution of TAG biosynthesis. The expression pattern of individual genes may be responsible for adaptation into multiple ecological niches. PMID:26721558

  5. High-Density Lipoprotein, Lecithin: Cholesterol Acyltransferase, and Atherosclerosis

    PubMed Central

    Ossoli, Alice; Pavanello, Chiara

    2016-01-01

    Epidemiological data clearly show the existence of a strong inverse correlation between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and the incidence of coronary heart disease. This relation is explained by a number of atheroprotective properties of HDL, first of all the ability to promote macrophage cholesterol transport. HDL are highly heterogeneous and are continuously remodeled in plasma thanks to the action of a number of proteins and enzymes. Among them, lecithin:cholesterol acyltransferase (LCAT) plays a crucial role, being the only enzyme able to esterify cholesterol within lipoproteins. LCAT is synthetized by the liver and it has been thought to play a major role in reverse cholesterol transport and in atheroprotection. However, data from animal studies, as well as human studies, have shown contradictory results. Increased LCAT concentrations are associated with increased HDL-C levels but not necessarily with atheroprotection. On the other side, decreased LCAT concentration and activity are associated with decreased HDL-C levels but not with increased atherosclerosis. These contradictory results confirm that HDL-C levels per se do not represent the functionality of the HDL system. PMID:27302716

  6. High-Density Lipoprotein, Lecithin: Cholesterol Acyltransferase, and Atherosclerosis.

    PubMed

    Ossoli, Alice; Pavanello, Chiara; Calabresi, Laura

    2016-06-01

    Epidemiological data clearly show the existence of a strong inverse correlation between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and the incidence of coronary heart disease. This relation is explained by a number of atheroprotective properties of HDL, first of all the ability to promote macrophage cholesterol transport. HDL are highly heterogeneous and are continuously remodeled in plasma thanks to the action of a number of proteins and enzymes. Among them, lecithin:cholesterol acyltransferase (LCAT) plays a crucial role, being the only enzyme able to esterify cholesterol within lipoproteins. LCAT is synthetized by the liver and it has been thought to play a major role in reverse cholesterol transport and in atheroprotection. However, data from animal studies, as well as human studies, have shown contradictory results. Increased LCAT concentrations are associated with increased HDL-C levels but not necessarily with atheroprotection. On the other side, decreased LCAT concentration and activity are associated with decreased HDL-C levels but not with increased atherosclerosis. These contradictory results confirm that HDL-C levels per se do not represent the functionality of the HDL system. PMID:27302716

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

    PubMed

    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. PMID:24850807

  8. Adiponutrin Functions as a Nutritionally Regulated Lysophosphatidic Acid Acyltransferase

    PubMed Central

    Kumari, Manju; Schoiswohl, Gabriele; Chitraju, Chandramohan; Paar, Margret; Cornaciu, Irina; Rangrez, Ashraf Y.; Wongsiriroj, Nuttaporn; Nagy, Harald M.; Ivanova, Pavlina T.; Scott, Sarah A.; Knittelfelder, Oskar; Rechberger, Gerald N.; Birner-Gruenberger, Ruth; Eder, Sandra; Brown, H. Alex; Haemmerle, Guenter; Oberer, Monika; Lass, Achim; Kershaw, Erin E.; Zimmermann, Robert; Zechner, Rudolf

    2012-01-01

    SUMMARY Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant. PMID:22560221

  9. Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

    PubMed Central

    Dunn, Briana J.; Khosla, Chaitan

    2013-01-01

    Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active ‘unnatural’ natural products. PMID:23720536

  10. Alcohol Alert

    MedlinePlus

    ... main content National Institute on Alcohol Abuse and Alcoholism (NIAAA) Main Menu Search Search form Search Alcohol & ... on a single aspect of alcohol abuse and alcoholism. Please click on the desired publication for full ...

  11. A close look at a ketosynthase from a trans-acyltransferase modular polyketide synthase

    PubMed Central

    Gay, Darren C.; Gay, Glen; Axelrod, Abram J.; Jenner, Matthew; Kohlhaas, Christoph; Kampa, Annette; Oldham, Neil J.; Piel, Jörn; Keatinge-Clay, Adrian T.

    2014-01-01

    SUMMARY The recently discovered trans-acyltransferase modular polyketide synthases catalyze the biosynthesis of a wide range of bioactive natural products in bacteria. Here we report the structure of the second ketosynthase from the bacillaene trans-acyltransferase polyketide synthase. This 1.95 Å-resolution structure provides the highest resolution view available of a modular polyketide synthase ketosynthase and reveals a flanking subdomain that is homologous to an ordered linker in cis-acyltransferase modular polyketide synthases. The structure of the cysteine-to-serine mutant of the ketosynthase acylated by its natural substrate provides high-resolution details of how a native polyketide intermediate is bound and helps explain the basis of ketosynthase substrate specificity. The substrate range of the ketosynthase was further investigated by mass spectrometry. PMID:24508341

  12. Alcoholism, Alcohol, and Drugs

    ERIC Educational Resources Information Center

    Rubin, Emanuel; Lieber, Charles S.

    1971-01-01

    Describes research on synergistic effects of alcohol and other drugs, particularly barbiturates. Proposes biochemical mechanisms to explain alcoholics' tolerance of other drugs when sober, and increased sensitivity when drunk. (AL)

  13. Membrane topology of murine glycerol-3-phosphate acyltransferase 2.

    PubMed

    Nakagawa, Tadahiko; Harada, Nagakatsu; Miyamoto, Aiko; Kawanishi, Yukiko; Yoshida, Masaki; Shono, Masayuki; Mawatari, Kazuaki; Takahashi, Akira; Sakaue, Hiroshi; Nakaya, Yutaka

    2012-02-17

    Glycerol-3-phosphate acyltransferase (GPAT) is a rate-limiting enzyme in mammalian triacylglycerol biosynthesis. GPAT is a target for the treatment of metabolic disorders associated with high lipid accumulation. Although the molecular basis for GPAT1 activation has been investigated extensively, the activation of other isoforms, such as GPAT2, is less well understood. Here the membrane topology of the GPAT2 protein was examined using an epitope-tag-based method. Exogenously expressed GPAT2 protein was present in the membrane fraction of transformed HEK293 cells even in the presence of Na(2)CO(3) (100 mM), indicating that GPAT2 is a membrane-bound protein. Trypsin treatment of the membrane fraction degraded the N-terminal (FLAG) and C-terminal (myc-epitope) protein tags of the GPAT2 protein. Bioinformatic analysis of the GPAT2 protein sequence indicated four hydrophobic sequences as potential membrane-spanning regions (TM1-TM4). Immunoblotting of the myc-epitope tag, which was inserted between each TM region of the GPAT2 protein, showed that the amino acid sequence between TM3 and TM4 was protected from trypsin digestion. These results suggest that the GPAT2 protein has two transmembrane segments and that the N-terminal and C-terminal regions of this protein face the cytoplasm. These results also suggest that the enzymatically active motifs I-III of the GPAT2 protein face the cytosol, while motif IV is within the membrane. It is expected that the use of this topological model of GPAT2 will be essential in efforts to elucidate the molecular mechanisms of GPAT2 activity in mammalian cells. PMID:22285183

  14. Topology of 1-Acyl-sn-glycerol-3-phosphate Acyltransferases SLC1 and ALE1 and Related Membrane-bound O-Acyltransferases (MBOATs) of Saccharomyces cerevisiae*

    PubMed Central

    Pagac, Martin; de la Mora, Hector Vazquez; Duperrex, Cécile; Roubaty, Carole; Vionnet, Christine; Conzelmann, Andreas

    2011-01-01

    In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed. PMID:21849510

  15. A Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Acyl Hydrolase Activities1[W][OA

    PubMed Central

    Vijayaraj, Panneerselvam; Jashal, Charnitkaur B.; Vijayakumar, Anitha; Rani, Sapa Hima; Venkata Rao, D.K.; Rajasekharan, Ram

    2012-01-01

    Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX4D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions. PMID:22915575

  16. Draft Genome Sequence of an Endophytic Actinoplanes Species, Encoding Uncommon trans-Acyltransferase Polyketide Synthases

    PubMed Central

    Centeno-Leija, Sara; Vinuesa, Pablo; Rodríguez-Peña, Karol; Trenado-Uribe, Miriam; Cárdenas-Conejo, Yair; Serrano-Posada, Hugo; Rodríguez-Sanoja, Romina

    2016-01-01

    Actinoplanes is an endophytic actinobacterium isolated from the medicinal plant Amphipterygium adstringens. The strain draft genome sequence reveals a gene cluster involved in the biosynthesis of a hybrid trans-acyltransferase (AT) polyketide, an unconventional bioactive metabolite never reported before in the genus Actinoplanes. PMID:27013046

  17. Structure-function analysis of diacylglycerol acyltransferase sequences for metabolic engineering and drug discovery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. Over-expression of DGATs increases TAG in plants. Therefore, unde...

  18. Structure-function analysis of diacylglycerol acyltransferase sequences from tung tree and 82 other Organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferase family (DGATs) catalyzes the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGATs esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA. Understanding the roles of DGATs will help to create transgenic plants with v...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGAT) are responsible for the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes, including DGAT1 and DGAT2 of tung tre...

  20. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants with value-added properties and provide clues for therapeutic intervention for obes...

  1. Castor diacylglycerol acyltransferase type1(DGAT1)displays greater activity with diricinolein than Arabidopsis DGAT1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil contains the hydroxy fatty acid ricinoleate as a major (90%) component. The diacylglycerol acyltransferase (DGAT) carries out the final reaction step in the biosynthesis of triacylglycerol, the principal constituent of seed oil, and has been considered to be the step that controls the oil...

  2. Expression of tung seed diacylglycerol acyltransferases (DGAT) in E. coli and yeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG, resist obesity, and/or lack milk secretion. Over-expression of the DGATs increases TAG content in seeds and other t...

  3. Alcoholism - resources

    MedlinePlus

    Resources - alcoholism ... The following organizations are good resources for information on alcoholism : Alcoholics Anonymous -- www.aa.org Al-Anon/Alateen -- www.al-anon.org/home National Institute on Alcohol ...

  4. Alcohol Alert

    MedlinePlus

    ... Us You are here Home » Alcohol Alert Alcohol Alert The NIAAA Alcohol Alert is a quarterly bulletin that disseminates important research ... text. To order single copies of select Alcohol Alerts, see ordering Information . To view publications in PDF ...

  5. Alcoholic neuropathy

    MedlinePlus

    Neuropathy - alcoholic; Alcoholic polyneuropathy ... The exact cause of alcoholic neuropathy is unknown. It likely includes both a direct poisoning of the nerve by the alcohol and the effect of poor nutrition ...

  6. Alcoholic ketoacidosis

    MedlinePlus

    Ketoacidosis - alcoholic ... Alcoholic ketoacidosis is caused by very heavy alcohol use. It most often occurs in a malnourished person ... Symptoms of alcoholic ketoacidosis include: Nausea and vomiting ... Changed level of alertness, which may lead to coma Confusion ...

  7. Alcohol Facts

    MedlinePlus

    ... raquo Alcohol Facts Alcohol Facts Listen Drinks like beer, malt liquor, wine, and hard liquor contain alcohol. Alcohol is the ingredient that gets you drunk. Hard liquor—such as whiskey, rum, or gin—has more ...

  8. Overexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli

    PubMed Central

    Yang, Lianqun; Zhang, Bin; Chen, Gao; Bi, Yuping

    2013-01-01

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

  9. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2.

    PubMed

    Cao, Heping; Chapital, Dorselyn C; Howard, O D; Deterding, Leesa J; Mason, Catherine B; Shockey, Jay M; Klasson, K Thomas

    2012-11-01

    Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may

  10. Functional Roles of Three Cutin Biosynthetic Acyltransferases in Cytokinin Responses and Skotomorphogenesis

    PubMed Central

    Chai, Juan; Zhou, Qin; Wang, Li; Hirnerová, Eva; Mrvková, Michaela; Novák, Ondřej; Guo, Guang-Qin

    2015-01-01

    Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis. PMID:25803274

  11. Alcohol Alert: Genetics of Alcoholism

    MedlinePlus

    ... and Reports » Alcohol Alert » Alcohol Alert Number 84 Alcohol Alert Number 84 Print Version The Genetics of ... immune defense system. Genes Encoding Enzymes Involved in Alcohol Breakdown Some of the first genes linked to ...

  12. Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene.

    PubMed Central

    Zou, J; Katavic, V; Giblin, E M; Barton, D L; MacKenzie, S L; Keller, W A; Hu, X; Taylor, D C

    1997-01-01

    A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs. PMID:9212466

  13. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis1s⃞

    PubMed Central

    Yuki, Koichi; Shindou, Hideo; Hishikawa, Daisuke; Shimizu, Takao

    2009-01-01

    Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and β-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis. PMID:19114731

  14. Evolutionarily Distinct BAHD N-Acyltransferases Are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice.

    PubMed

    Peng, Meng; Gao, Yanqiang; Chen, Wei; Wang, Wensheng; Shen, Shuangqian; Shi, Jian; Wang, Cheng; Zhang, Yu; Zou, Li; Wang, Shouchuang; Wan, Jian; Liu, Xianqing; Gong, Liang; Luo, Jie

    2016-07-01

    Phenolamides (PAs) are specialized (secondary) metabolites mainly synthesized by BAHD N-acyltransferases. Here, we report metabolic profiling coupled with association and linkage mapping of 11 PAs in rice (Oryza sativa). We identified 22 loci affecting PAs in leaves and 16 loci affecting PAs in seeds. We identified eight BAHD N-acyltransferases located on five chromosomes with diverse specificities, including four aromatic amine N-acyltransferases. We show that genetic variation in PAs is determined, at least in part, by allelic variation in the tissue specificity of expression of the BAHD genes responsible for their biosynthesis. Tryptamine hydroxycinnamoyl transferase 1/2 (Os-THT1/2) and tryptamine benzoyl transferase 1/2 (Os-TBT1/2) were found to be bifunctional tryptamine/tyramine N-acyltransferases. The specificity of Os-THT1 and Os-TBT1 for agmatine involved four tandem arginine residues, which have not been identified as specificity determinants for other plant BAHD transferases, illustrating the versatility of plant BAHD transferases in acquiring new acyl acceptor specificities. With phylogenetic analysis, we identified both divergent and convergent evolution of N-acyltransferases in plants, and we suggest that the BAHD family of tryptamine/tyramine N-acyltransferases evolved conservatively in monocots, especially in Gramineae. Our work demonstrates that omics-assisted gene-to-metabolite analysis provides a useful tool for bulk gene identification and crop genetic improvement. PMID:27354554

  15. Evolutionarily Distinct BAHD N-Acyltransferases Are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice[OPEN

    PubMed Central

    Peng, Meng; Chen, Wei; Wang, Wensheng; Shen, Shuangqian; Shi, Jian; Wang, Cheng; Zhang, Yu; Zou, Li; Wang, Shouchuang; Wan, Jian; Liu, Xianqing; Gong, Liang; Luo, Jie

    2016-01-01

    Phenolamides (PAs) are specialized (secondary) metabolites mainly synthesized by BAHD N-acyltransferases. Here, we report metabolic profiling coupled with association and linkage mapping of 11 PAs in rice (Oryza sativa). We identified 22 loci affecting PAs in leaves and 16 loci affecting PAs in seeds. We identified eight BAHD N-acyltransferases located on five chromosomes with diverse specificities, including four aromatic amine N-acyltransferases. We show that genetic variation in PAs is determined, at least in part, by allelic variation in the tissue specificity of expression of the BAHD genes responsible for their biosynthesis. Tryptamine hydroxycinnamoyl transferase 1/2 (Os-THT1/2) and tryptamine benzoyl transferase 1/2 (Os-TBT1/2) were found to be bifunctional tryptamine/tyramine N-acyltransferases. The specificity of Os-THT1 and Os-TBT1 for agmatine involved four tandem arginine residues, which have not been identified as specificity determinants for other plant BAHD transferases, illustrating the versatility of plant BAHD transferases in acquiring new acyl acceptor specificities. With phylogenetic analysis, we identified both divergent and convergent evolution of N-acyltransferases in plants, and we suggest that the BAHD family of tryptamine/tyramine N-acyltransferases evolved conservatively in monocots, especially in Gramineae. Our work demonstrates that omics-assisted gene-to-metabolite analysis provides a useful tool for bulk gene identification and crop genetic improvement. PMID:27354554

  16. Characterization of an Acyltransferase Capable of Synthesizing Benzylbenzoate and Other Volatile Esters in Flowers and Damaged Leaves of Clarkia breweri1

    PubMed Central

    D'Auria, John C.; Chen, Feng; Pichersky, Eran

    2002-01-01

    A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile. PMID:12226525

  17. Alcoholic ketoacidosis

    MedlinePlus

    ... attention improves the overall outlook. How severe the alcoholism is, and the presence of liver disease or ... A.M. Editorial team. Related MedlinePlus Health Topics Alcoholism and Alcohol Abuse Browse the Encyclopedia A.D. ...

  18. Alcohol withdrawal

    MedlinePlus

    ... counseling to discuss the long-term issue of alcoholism Testing and treatment for other medical problems linked ... following organizations are good resources for information on alcoholism: Alcoholics Anonymous -- www.aa.org Al-Anon/Alateen -- ...

  19. Alcoholic neuropathy

    MedlinePlus

    ... objects in the shoes Guarding the extremities to prevent injury from pressure Alcohol must be stopped to prevent the damage from ... The only way to prevent alcoholic neuropathy is not to drink excessive amounts of alcohol.

  20. The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

    PubMed Central

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

    2014-01-01

    Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates. PMID:25331437

  1. Isolation of Acyl-CoA:cholesterol acyltransferase inhibitor from Persicaria vulgaris.

    PubMed

    Song, Hye Young; Rho, Mun-Chual; Lee, Seung Woong; Kwon, Oh Eok; Chang, Young-Duck; Lee, Hyun Sun; Kim, Young-Kook

    2002-09-01

    In the course of our search for Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors from natural sources, a new type of ACAT inhibitor was isolated from the methanol extract of Persicaria vulgaris. On the basis of spectral evidence, the structure of the active compound was identified as pheophorbide A. Pheophorbide A inhibited ACAT activity with an IC 50 value of 1.1 microg/ml in an enzyme assay using rat liver microsomes with a dose dependent fashion. PMID:12357403

  2. Identification of a broad family of lipid A late acyltransferases with non-canonical substrate specificity

    PubMed Central

    Rubin, Erica J.; O’Brien, John P.; Ivanov, Petko L.; Brodbelt, Jennifer S.; Trent, M. Stephen

    2014-01-01

    Summary Most gram-negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine-step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3′-linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyzes addition of a C12:0 or C14:0 acyl chain to the 3′-linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologs in Campylobacter jejuni and Wolinella succinogenes can act before the 2′ secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. PMID:24372821

  3. Characterization of monoacylglycerol acyltransferase 2 inhibitors by a novel probe in binding assays.

    PubMed

    Ma, Zhengping; Chao, Hannguang J; Turdi, Huji; Hangeland, Jon J; Friends, Todd; Kopcho, Lisa M; Lawrence, R Michael; Cheng, Dong

    2016-05-15

    Monoacylglycerol acyltransferase 2 (MGAT2) is a membrane-bound lipid acyltransferase that catalyzes the formation of diacylglycerol using monoacylglycerol and fatty acyl CoA as substrates. MGAT2 is important for intestinal lipid absorption and is an emerging target for the treatment of metabolic diseases. In the current study, we identified and characterized four classes of novel MGAT2 inhibitors. We established both steady state and kinetic binding assay protocols using a novel radioligand, [(3)H]compound A. Diverse chemotypes of MGAT2 inhibitors were found to compete binding of [(3)H]compound A to MGAT2, indicating the broad utility of [(3)H]compound A for testing various classes of MGAT2 inhibitors. In the dynamic binding assays, the kinetic values of MGAT2 inhibitors such as Kon, Koff, and T1/2 were systematically defined. Of particular value, the residence times of inhibitors on MGAT2 enzyme were derived. We believe that the identification of novel classes of MGAT2 inhibitors and the detailed kinetic characterization provide valuable information for the identification of superior candidates for in vivo animal and clinical studies. The current work using a chemical probe to define inhibitory kinetics can be broadly applied to other membrane-bound acyltransferases. PMID:26925857

  4. The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

    PubMed

    Gay, Darren C; Wagner, Drew T; Meinke, Jessica L; Zogzas, Charles E; Gay, Glen R; Keatinge-Clay, Adrian T

    2016-03-01

    Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. PMID:26724270

  5. Glucose Polyester Biosynthesis. Purification and Characterization of a Glucose Acyltransferase1

    PubMed Central

    Li, Alice X.; Eannetta, Nancy; Ghangas, Gurdev S.; Steffens, John C.

    1999-01-01

    Glandular trichomes of the wild tomato species Lycopersicon pennellii secrete 2,3,4-O-tri-acyl-glucose (-Glc), which contributes to insect resistance. A Glc acyltransferase catalyzes the formation of diacyl-Glc by disproportionating two equivalents of 1-O-acyl-β-Glc, a high-energy molecule formed by a UDP-Glc dependent reaction. The acyltransferase was purified 4,900-fold from L. pennellii leaves by polyethylene glycol fractionation, diethylaminoethyl chromatography, concanavalin A affinity chromatography, and chromatofocusing. The acyltransferase possesses an isoelectric point of 4.8, a relative molecular mass around 110 kD, and is composed of 34- and 24-kD polypeptides as a heterotetramer. The 34- and 24-kD proteins were partially sequenced. The purified enzyme catalyzes both the disproportionation of 1-O-acyl-β-Glcs to generate 1,2-di-O-acyl-β-Glc and anomeric acyl exchange between 1-O-acyl-β-Glc and Glc. PMID:10517836

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

    PubMed

    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. PMID:27506614

  7. Alcoholism and Alcohol Abuse

    MedlinePlus

    ... increase the risk of certain cancers. It can cause damage to the liver, brain, and other organs. Drinking during pregnancy can harm your baby. Alcohol also increases the risk of death from car crashes, injuries, homicide, and suicide. If you want to stop drinking, there is ...

  8. Metabolic engineering of Escherichia coli for production of biodiesel from fatty alcohols and acetyl-CoA.

    PubMed

    Guo, Daoyi; Pan, Hong; Li, Xun

    2015-09-01

    Microbial production of biodiesel from renewable feedstock has attracted intensive attention. Biodiesel is known to be produced from short-chain alcohols and fatty acyl-CoAs through the expression of wax ester synthase/fatty acyl-CoA: diacylglycerol acyltransferase that catalyzes the esterification of short-chain alcohols and fatty acyl-CoAs. Here, we engineered Escherichia coli to produce various fatty alcohol acetate esters, which depend on the expression of Saccharomyces cerevisiae alcohol acetyltransferase ATF1 that catalyzes the esterification of fatty alcohols and acetyl-CoA. The fatty acid biosynthetic pathways generate fatty acyl-ACPs, fatty acyl-CoAs, or fatty acids, which can be converted to fatty alcohols by fatty acyl-CoA reductase, fatty acyl-ACP reductase, or carboxylic acid reductase, respectively. This study showed the biosynthesis of biodiesel from three fatty acid biosynthetic pathway intermediates. PMID:26205521

  9. Alcohol Calorie Calculator

    MedlinePlus

    ... Alcohol Calorie Calculator Weekly Total 0 Calories Alcohol Calorie Calculator Find out the number of beer and ... Calories College Alcohol Policies Interactive Body Calculators Alcohol Calorie Calculator Alcohol Cost Calculator Alcohol BAC Calculator Alcohol ...

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

    PubMed

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

    2007-11-13

    Cutin and suberin are the two major lipid-based polymers of plants. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs and which is often the structure first encountered by phytopathogens. Suberin contributes to the control of diffusion of water and solutes across internal root tissues and in periderms. The enzymes responsible for assembly of the cutin polymer are largely unknown. We have identified two Arabidopsis acyltransferases essential for cutin biosynthesis, glycerol-3-phosphate acyltransferase (GPAT) 4 and GPAT8. Double knockouts gpat4/gpat8 were strongly reduced in cutin and were less resistant to desiccation and to infection by the fungus Alternaria brassicicola. They also showed striking defects in stomata structure including a lack of cuticular ledges between guard cells, highlighting the importance of cutin in stomatal biology. Overexpression of GPAT4 or GPAT8 in Arabidopsis increased the content of C16 and C18 cutin monomers in leaves and stems by 80%. In order to modify cutin composition, the acyltransferase GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two enzymes associated with suberin biosynthesis, were overexpressed. When both enzymes were overexpressed together the epidermal polyesters accumulated new C20 and C22 omega-hydroxyacids and alpha,omega-diacids typical of suberin, and the fine structure and water-barrier function of the cuticle were altered. These results identify GPATs as partners of fatty acyl oxidases in lipid polyester synthesis and indicate that their cooverexpression provides a strategy to probe the role of cutin composition and quantity in the function of plant cuticles. PMID:17991776

  11. CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase.

    PubMed

    Montero-Moran, Gabriela; Caviglia, Jorge M; McMahon, Derek; Rothenberg, Alexis; Subramanian, Vidya; Xu, Zhi; Lara-Gonzalez, Samuel; Storch, Judith; Carman, George M; Brasaemle, Dawn L

    2010-04-01

    Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an alpha/beta-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids. PMID:19801371

  12. Some kinetic properties of plasma lecithin-cholesterol acyltransferase in hyper-alphalipoproteinemia in man

    SciTech Connect

    Nikiforova, A.A.; Alksnis, E.G.; Ivanova, E.M.

    1985-07-01

    The aim of this investigation was to study some kinetic properties of lecithin-cholesterol acyltransferase (LCAT) in the blood plasma of patients with hyper-alpha-lipoproteinemia, enabling the presence of LCAT isozymes in the blood to be detected. The velocity of the LCAT reaction was judged by determining labeled CHE formed from /sup 14/C-nonesterified CH and lecithin of HDL on incubation of the latter with the enzyme. Dependence of the velocity of the LCAT reaction on concentration of substrate (nonesterified HDL cholesterol) in four subjects with hyper-alpha-lipoproteinemia is shown.

  13. Inhibition by acetyl-CoA of hepatic carnitine acyltransferase and fatty acid oxidation.

    PubMed Central

    McCormick, K; Notar-Francesco, V J; Sriwatanakul, K

    1983-01-01

    At micromolar concentrations, acetyl-CoA inhibited hepatic carnitine acyltransferase activity and mitochondrial fatty acid oxidation. The inhibitory effects were not nearly as potent on a molar basis as those of malonyl-CoA; nevertheless, the cytosolic concentrations of acetyl-CoA, as yet unknown, may be sufficient (greater than 30 microM) to curtail appreciably the mitochondrial transfer of long-chain acyl-CoA units and fatty acid oxidation. Hence acetyl-CoA may also partially regulate hepatic ketogenesis. PMID:6661211

  14. Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii.

    PubMed

    Hung, Chun-Hsien; Ho, Ming-Yang; Kanehara, Kazue; Nakamura, Yuki

    2013-08-01

    Algal triacylglycerol biosynthesis is of increasing interest for potential biodiesel production. A model microalga, Chlamydomonas, has multiple isoforms of diacylglycerol acyltransferase type 2 (DGTT) catalyzing the final step of triacylglycerol biosynthesis; however, the functions of the isoforms are poorly understood. Here, we performed heterologous complementation assay of Chlamydomonas DGTT1 to 4 in a yeast mutant defective in triacylglycerol biosynthesis. DGTT1, 2 and 3 but not 4 complemented the phenotype, including triacylglycerol levels. Interestingly, complementation by DGTT2 increased triacylglycerol content by 9-fold. PMID:23770092

  15. Selectivity of pyripyropene derivatives in inhibition toward acyl-CoA:cholesterol acyltransferase 2 isozyme.

    PubMed

    Ohshiro, Taichi; Ohte, Satoshi; Matsuda, Daisuke; Ohtawa, Masaki; Nagamitsu, Tohru; Sunazuka, Toshiaki; Harigaya, Yoshihiro; Rudel, Lawrence L; Omura, Satoshi; Tomoda, Hiroshi

    2008-08-01

    Selectivity of 96 semisynthetic derivatives prepared from fungal pyripyropene A, originally isolated as a potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), toward ACAT1 and ACAT2 isozymes was investigated in the cell-based assay using ACAT1- and ACAT2-expressing CHO cells. Eighteen derivatives including PR-71 (7-O-isocaproyl derivative) showed much more potent ACAT2 inhibition (IC50: 6.0 to 62 nM) than pyripyropene A (IC50: 70 nM). Among them, however, natural pyripyropene A showed the highest selectivity toward ACAT2 with a selectivity index (SI) of >1000, followed by PR-71 (SI, 667). PMID:18997389

  16. Propyl alcohol

    MedlinePlus

    Rubbing alcohol Alcohol swabs Skin and hair products Nail polish remover Note: This list may not be all ... number will let you talk to experts in poisoning. They will give you further instructions. This is ...

  17. Alcoholic hallucinosis.

    PubMed

    Bhat, Pookala S; Ryali, Vssr; Srivastava, Kalpana; Kumar, Shashi R; Prakash, Jyoti; Singal, Ankit

    2012-07-01

    Alcoholic hallucinosis is a rare complication of chronic alcohol abuse characterized by predominantly auditory hallucinations that occur either during or after a period of heavy alcohol consumption. Bleuler (1916) termed the condition as alcohol hallucinosis and differentiated it from Delirium Tremens. Usually it presents with acoustic verbal hallucinations, delusions and mood disturbances arising in clear consciousness and sometimes may progress to a chronic form mimicking schizophrenia. One such case with multimodal hallucinations in a Defence Service Corps soldier is presented here. PMID:24250051

  18. Alcohol Abuse

    ERIC Educational Resources Information Center

    O'Farrell, Timothy J.; Fals-Stewart, William

    2003-01-01

    We received 38 controlled studies of marital and family therapy (MFT) in alcoholism treatment. We conclude that, when the alcoholic is unwilling to seek help, MFT is effective in helping the family cope better and motivating alcoholics to enter treatment. Specifically, (a) Al-Anon facilitation and referral help family members cope better; (b)…

  19. Diacylglycerol O-Acyltransferase Type-1 Synthesizes Retinyl Esters in the Retina and Retinal Pigment Epithelium

    PubMed Central

    Kaylor, Joanna J.; Radu, Roxana A.; Bischoff, Nicholas; Makshanoff, Jacob; Hu, Jane; Lloyd, Marcia; Eddington, Shannan; Bianconi, Tran; Bok, Dean; Travis, Gabriel H.

    2015-01-01

    Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1 -/- mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1 -/- mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE’s) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1 -/- mice. Further, total retinyl-ester synthase activity was reduced in both dgat1 -/- retina and RPE. PMID:25974161

  20. Role of HXXXD-motif/BAHD acyltransferases in the biosynthesis of extracellular lipids.

    PubMed

    Molina, Isabel; Kosma, Dylan

    2015-04-01

    Terrestrial plants have evolved specific adaptations to preserve water and protect themselves from their environment. Such adaptations range from secondary metabolites and specialized structures that conduct water and nutrients, to cell wall modifications (i.e., cuticle and suberin) that prevent dehydration and provide a physical barrier to pathogens. Both the plant cuticle and suberized cell walls contain a lipid polymer framework embedded with waxes, and constitute a promising target for controlled genetic modification to improve desirable agronomic traits. Recent advances in genomic and molecular techniques coupled with the development of robust analytical methods have accelerated progress in comprehending these intractable lipid polymers. Gene products characterized in the wax, cutin and suberin pathways include a subset of HXXXD/BAHD family enzymes that catalyze acyl transfer reactions between CoA-activated hydroxycinnamic acid derivatives and hydroxylated aliphatics. This review highlights our current understanding of HXXXD/BAHD acyltransferases in extracellular lipid biosynthesis and discusses the chemical, ultrastructural and physiological ramifications of impairing the expression of BAHD acyltransferase-encoding genes related to cutin and suberin synthesis. PMID:25510356

  1. Architectural Organization of the Metabolic Regulatory Enzyme Ghrelin O-Acyltransferase*

    PubMed Central

    Taylor, Martin S.; Ruch, Travis R.; Hsiao, Po-Yuan; Hwang, Yousang; Zhang, Pingfeng; Dai, Lixin; Huang, Cheng Ran Lisa; Berndsen, Christopher E.; Kim, Min-Sik; Pandey, Akhilesh; Wolberger, Cynthia; Marmorstein, Ronen; Machamer, Carolyn; Boeke, Jef D.; Cole, Philip A.

    2013-01-01

    Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, facilitated these experiments. The MBOAT family invariant residue His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately advance the discovery of selective inhibitors for these enzymes. PMID:24045953

  2. Human 1-Acylglycerol-3-phosphate O-Acyltransferase Isoforms 1 and 2

    PubMed Central

    Agarwal, Anil K.; Sukumaran, Suja; Cortés, Víctor A.; Tunison, Katie; Mizrachi, Dario; Sankella, Shireesha; Gerard, Robert D.; Horton, Jay D.; Garg, Abhimanyu

    2011-01-01

    Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2−/− mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2−/− mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2−/− mice. PMID:21873652

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  4. Regulation of acyltransferase activity in immature maize embryos by abscisic acid and the osmotic environment.

    PubMed Central

    Pacheco-Moisés, F; Valencia-Turcotte, L; Altuzar-Martínez, M; Rodríguez-Sotres, R

    1997-01-01

    Maize (Zes mays L.) embryos, isolated from the developing seed and incubated in dilute buffer, show reduced triacylglycerol (TAG) synthesis, and accumulation stops after 24 h. Synthesis and accumulation can be maintained at high levels if the incubation medium contains abscisic acid (ABA) and/or a high osmotic concentration. Radiolabeled free fatty acids accumulate at higher levels in embryos that contain less TAG, and acetyl coenzyme A carboxylase activity remains essentially unchanged under all of the conditions tested. In contrast, the activities of the acyltransferases required for TAG synthesis remain high only in embryos incubated with ABA and/or a high osmotic concentration. Dose-response curves showed that 4 microM of ABA or mannitol at -1.0 MPa elicits a full response; both values are within the range considered to be physiological. The TAG synthesis capacity and discylglycerol acyltransferase activity lost by pretreatment of the embryos can be restored by re-exposure to ABA or high osmoticum. Germination is not involved because isolated scutellum halves showed the same changes in enzyme activity found in the whole embryo but did not germinate. Our results provide direct evidence for the regulation of TAG-synthesizing activities in maize embryos by ABA and the osmotic potential of the environment. PMID:9232885

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

    PubMed Central

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

    2006-01-01

    Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4′-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB. PMID:16735476

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

    PubMed

    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. PMID:23103975

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

    PubMed Central

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

    2016-01-01

    C-4 hydroxyethyl branched octoses have been observed in polysaccharides of several genera of gram negative bacteria and in various antibiotics produced by gram positive bacteria. The C-4 hydroxyethyl branch was proposed to be converted from C-4 acetyl branch by an uncharacterized ketoreduction step. Paulomycins (PAUs) are glycosylated antibiotics with potent inhibitory activity against gram positive bacteria and are structurally defined by its unique C-4′ hydroxyethyl branched paulomycose moiety. A novel aldo-keto-reductase, Pau7 was characterized as the enzyme catalyzing the stereospecific ketoreduction of 7′-keto of PAU E (1) to give the C-4′ hydroxyethyl branched paulomycose moiety of PAU F (2). An acyltransferase Pau6 further decorates the C-4′ hydroxyethyl branch of paulomycose moiety of 2 by attaching various fatty acyl chains to 7′-OH to generate diverse PAUs. In addition, another acyltransferase Pau24 was proposed to be responsible for the 13-O-acetylation of PAUs. PMID:26877148

  8. Facts about Alcohol and Alcoholism.

    ERIC Educational Resources Information Center

    Hall, Leonard C.

    Recognition of alcoholism as a treatable illness is a result of public education based on scientific facts. This publication, a digest of a more detailed survey of research about drinking and alcoholism, presents information about alcohol and its effects on individuals and society. It provides facts about the short-term and long-term effects of…

  9. Alcoholic cardiomyopathy

    PubMed Central

    Guzzo-Merello, Gonzalo; Cobo-Marcos, Marta; Gallego-Delgado, Maria; Garcia-Pavia, Pablo

    2014-01-01

    Alcohol is the most frequently consumed toxic substance in the world. Low to moderate daily intake of alcohol has been shown to have beneficial effects on the cardiovascular system. In contrast, exposure to high levels of alcohol for a long period could lead to progressive cardiac dysfunction and heart failure. Cardiac dysfunction associated with chronic and excessive alcohol intake is a specific cardiac disease known as alcoholic cardiomyopathy (ACM). In spite of its clinical importance, data on ACM and how alcohol damages the heart are limited. In this review, we evaluate available evidence linking excessive alcohol consumption with heart failure and dilated cardiomyopathy. Additionally, we discuss the clinical presentation, prognosis and treatment of ACM. PMID:25228956

  10. Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles inacyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for se...

  11. The Glycerol-3-Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei.

    PubMed

    Patel, Nipul; Pirani, Karim A; Zhu, Tongtong; Cheung-See-Kit, Melanie; Lee, Sungsu; Chen, Daniel G; Zufferey, Rachel

    2016-09-01

    Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol-3-phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol-3-phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl-CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N-terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol-3-phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI-anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence. PMID:26909872

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

    PubMed

    Zhang, Donghui; Jasieniecka-Gazarkiewicz, Katarzyna; Wan, Xia; Luo, Ling; Zhang, Yinbo; Banas, Antoni; Jiang, Mulan; Gong, Yangmin

    2015-01-01

    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

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

    PubMed Central

    Wan, Xia; Luo, Ling; Zhang, Yinbo; Banas, Antoni; Jiang, Mulan; Gong, Yangmin

    2015-01-01

    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. Overview of Alcohol Consumption

    MedlinePlus

    ... Search Alcohol & Your Health Overview of Alcohol Consumption Alcohol's Effects on the Body Alcohol Use Disorder Fetal Alcohol ... other questions about alcohol. Here’s what we know: Alcohol’s effects vary from person to person, depending on a ...

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-10-01

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

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

    PubMed

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

    2015-01-01

    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. PMID:25727495

  18. Identification of the active-site serine in human lecithin: cholesterol acyltransferase

    SciTech Connect

    Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.

    1987-05-01

    Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.

  19. Catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides

    SciTech Connect

    Park, Y.B.; Yueksel, U.G.; Gracy, R.W.; Lacko, A.G.

    1987-02-27

    Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with (/sup 3/H)-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. BacMam production of active recombinant lecithin-cholesterol acyltransferase: Expression, purification and characterization.

    PubMed

    Romanow, William G; Piper, Derek E; Fordstrom, Preston; Thibault, Stephen; Zhou, Mingyue; Walker, Nigel P C

    2016-09-01

    Lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed. PMID:26363122

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25727495

  4. Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.

    PubMed Central

    Fernández, F J; Gutierrez, S; Velasco, J; Montenegro, E; Marcos, A T; Martín, J F

    1994-01-01

    Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits. Images PMID:7519594

  5. Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba) 1

    PubMed Central

    Cao, Yi-zhi; Oo, Khaik-Cheang; Huang, Anthony H. C.

    1990-01-01

    Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl2, ZnCl2, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [14C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering. PMID:16667817

  6. Homologous yeast lipases/acyltransferases exhibit remarkable cold-active properties.

    PubMed

    Neang, Pisey M; Subileau, Maeva; Perrier, Véronique; Dubreucq, Eric

    2014-11-01

    Lipases/acyltransferases catalyse acyltransfer to various nucleophiles preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (a w >0.9). Characterization of hydrolysis and acyltransfer activities in a large range of temperature (5 to 80 °C) of secreted recombinant homologous lipases of the Pseudozyma antarctica lipase A superfamily (CaLA) expressed in Pichia pastoris, enlighten the exceptional cold-activity of two remarkable lipases/acyltransferases: CpLIP2 from Candida parapsilosis and CtroL4 from Candida tropicalis. The activation energy of the reactions catalysed by CpLIP2 and CtroL4 was 18-23 kJ mol(-1) for hydrolysis and less than 15 kJ mol(-1) for transesterification between 5 and 35 °C, while it was respectively 43 and 47 kJ mol(-1) with the thermostable CaLA. A remarkable consequence is the high rate of the reactions catalysed by CpLIP2 and CtroL4 at very low temperatures, with CpLIP2 displaying at 5 °C 65 % of its alcoholysis activity and 45 % of its hydrolysis activity at 30 °C. These results suggest that, within the CaLA superfamily and its homologous subgroups, common structural determinants might allow both acyltransfer and cold-active properties. Such biocatalysts are of great interest for the efficient synthesis or functionalization of temperature-sensitive lipid derivatives, or more generally to lessen the environmental impact of biocatalytic processes. PMID:24770385

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

    PubMed

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

    2016-02-01

    Dietary triglycerides (TG) are absorbed by the enterocytes of the small intestine after luminal hydrolysis into monacylglycerol and fatty acids. Before secretion on chylomicrons, these lipids are reesterified into TG, primarily through the monoacylglycerol pathway. However, targeted deletion of the primary murine monoacylglycerol acyltransferase does not quantitatively affect lipid absorption, suggesting the existence of alternative pathways. Therefore, we investigated the role of the glycerol 3-phosphate pathway in dietary lipid absorption. The expression of glycerol-3-phosphate acyltransferase (GPAT3) was examined throughout the small intestine. To evaluate the role for GPAT3 in lipid absorption, mice harboring a disrupted GPAT3 gene (Gpat3(-/-)) were subjected to an oral lipid challenge and fed a Western-type diet to characterize the role in lipid and cholesterol homeostasis. Additional mechanistic studies were performed in primary enterocytes. GPAT3 was abundantly expressed in the apical surface of enterocytes in the small intestine. After an oral lipid bolus, Gpat3(-/-) mice exhibited attenuated plasma TG excursion and accumulated lipid in the enterocytes. Electron microscopy studies revealed a lack of lipids in the lamina propria and intercellular space in Gpat3(-/-) mice. Gpat3(-/-) enterocytes displayed a compensatory increase in the synthesis of phospholipid and cholesteryl ester. When fed a Western-type diet, hepatic TG and cholesteryl ester accumulation was significantly higher in Gpat3(-/-) mice compared with the wild-type mice accompanied by elevated levels of alanine aminotransferase, a marker of liver injury. Dysregulation of bile acid metabolism was also evident in Gpat3-null mice. These studies identify GPAT3 as a novel enzyme involved in intestinal lipid metabolism. PMID:26644473

  8. Comparative gene identification-58 (CGI-58) promotes autophagy as a putative lysophosphatidylglycerol acyltransferase.

    PubMed

    Zhang, Jun; Xu, Dan; Nie, Jia; Han, Ruili; Zhai, Yonggong; Shi, Yuguang

    2014-11-21

    CGI-58 is a lipid droplet-associated protein that, when mutated, causes Chanarin-Dorfman syndrome in humans, which is characterized by excessive storage of triglyceride in various tissues. However, the molecular mechanisms underlying the defect remain elusive. CGI-58 was previously reported to catalyze the resynthesis of phosphatidic acid as a lysophosphatidic acid acyltransferase. In addition to triglyceride, phosphatidic acid is also used a substrate for the synthesis of various mitochondrial phospholipids. In this report, we investigated the propensity of CGI-58 in the remodeling of various phospholipids. We found that the recombinant CGI-58 overexpressed in mammalian cells or purified from Sf9 insect cells catalyzed efficiently the reacylation of lysophosphatidylglycerol to phosphatidylglycerol (PG), which requires acyl-CoA as the acyl donor. In contrast, the recombinant CGI-58 was devoid of acyltransferase activity toward other lysophospholipids. Accordingly, overexpression and knockdown of CGI-58 adversely affected the endogenous PG level in C2C12 cells. PG is a substrate for the synthesis of cardiolipin, which is required for mitochondrial oxidative phosphorylation and mitophagy. Consequently, overexpression and knockdown of CGI-58 adversely affected autophagy and mitophagy in C2C12 cells. In support for a key role of CGI-58 in mitophagy, overexpression of CGI-58 significantly stimulated mitochondrial fission and translocation of PINK1 to mitochondria, key steps involved in mitophagy. Furthermore, overexpression of CGI-58 promoted mitophagic initiation through activation of 5'-AMP-activated protein kinase and inhibition of mTORC1 mammalian target of rapamycin complex 1 signaling, the positive and negative regulators of autophagy, respectively. Together, these findings identified novel molecular mechanisms by which CGI-58 regulates lipid homeostasis, because defective autophagy is implicated in dyslipidemia and fatty liver diseases. PMID:25315780

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

    PubMed

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

    2013-07-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) catalyzes acylation of lysophosphatidylcholine (lysoPtdCho) to produce phosphatidylcholine (PtdCho), the main phospholipid in cellular membranes. This reaction is a key component of the acyl-editing process, involving recycling of the fatty acids (FA) mainly at the sn-2 position of PtdCho. Growing evidences indicate that the LPCAT reaction controls the direct entry of newly synthesized FA into PtdCho and, at least in some plant species, it has an important impact on the synthesis and composition of triacylglycerols. Here we describe the molecular characterization of the single LPCAT gene found in the genome of Ricinus communis (RcLPCAT) that is homologous to LPCAT genes of the MBOAT family previously described in Arabidopsis and Brassica. RcLPCAT is ubiquitously expressed in all organs of the castor plant. Biochemical properties have been studied by heterologous expression of RcLPCAT in the ale1 yeast mutant, defective in lysophospholipid acyltransferase activity. RcLPCAT preferentially acylates lysoPtdCho against other lysophospholipids (lysoPL) and does not discriminates the acyl chain in the acceptor, displaying a strong activity with alkyl lysoPL. Regarding the acyl-CoA donor, RcLPCAT uses monounsaturated fatty acid thioesters, such as oleoyl-CoA (18:1-CoA), as preferred donors, while it has a low activity with saturated fatty acids and shows a poor utilization of ricinoleoyl-CoA (18:1-OH-CoA). These characteristics are discussed in terms of a possible role of RcLPCAT in regulating the entry of FA into PtdCho and the exclusion from the membranes of the hydroxylated FA. PMID:23700249

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

    SciTech Connect

    Joo, Sang Hoon; Chung, Hak Suk; Raetz, Christian R.H.; Garrett, Teresa A.

    2012-08-31

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

  11. Alcohol Energy Drinks

    MedlinePlus

    ... Home / About Addiction / Alcohol / Alcohol Energy Drinks Alcohol Energy Drinks Read 14635 times font size decrease font size increase font size Print Email Alcohol energy drinks (AEDs) or Caffeinated alcoholic beverages (CABs) are ...

  12. Alcohol Energy Drinks

    MedlinePlus

    ... Home / About Addiction / Alcohol / Alcohol Energy Drinks Alcohol Energy Drinks Read 17728 times font size decrease font size increase font size Print Email Alcohol energy drinks (AEDs) or Caffeinated alcoholic beverages (CABs) are ...

  13. Alcohol during Pregnancy

    MedlinePlus

    ... Home > Pregnancy > Is it safe? > Alcohol during pregnancy Alcohol during pregnancy E-mail to a friend Please ... and fetal alcohol spectrum disorders. How does drinking alcohol during pregnancy affect your baby's health? Drinking alcohol ...

  14. Cloning and identification of the human LPAAT-zeta gene, a novel member of the lysophosphatidic acid acyltransferase family.

    PubMed

    Li, Dan; Yu, Long; Wu, Hai; Shan, Yuxi; Guo, Jinhu; Dang, Yongjun; Wei, Youheng; Zhao, Shouyuan

    2003-01-01

    Lysophosphatidic acid (LPA) is a naturally occurring component of phospholipid and plays a critical role in the regulation of many physiological and pathophysiological processes including cell growth, survival, and pro-angiogenesis. LPA is converted to phosphatidic acid by the action of lysophosphatidic acid acyltransferase (LPAAT). Five members of the LPAAT gene family have been detected in humans to date. Here, we report the identification of a novel LPAAT member, which is designated as LPAAT-zeta. LPAAT-zeta was predicted to encode a protein consisting of 456 amino acid residues with a signal peptide sequence and the acyltransferase domain. Northern blot analysis showed that LPAAT-zeta was ubiquitously expressed in all 16 human tissues examined, with levels in the skeletal muscle, heart, and testis being relatively high and in the lung being relatively low. The human LPAAT-zeta gene consisted of 13 exons and is positioned at chromosome 8p11.21. PMID:12938015

  15. Alcohol conversion

    DOEpatents

    Wachs, Israel E.; Cai, Yeping

    2002-01-01

    Preparing an aldehyde from an alcohol by contacting the alcohol in the presence of oxygen with a catalyst prepared by contacting an intimate mixture containing metal oxide support particles and particles of a catalytically active metal oxide from Groups VA, VIA, or VIIA, with a gaseous stream containing an alcohol to cause metal oxide from the discrete catalytically active metal oxide particles to migrate to the metal oxide support particles and to form a monolayer of catalytically active metal oxide on said metal oxide support particles.

  16. A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol.

    PubMed

    Yang, Weili; Pollard, Mike; Li-Beisson, Yonghua; Beisson, Fred; Feig, Michael; Ohlrogge, John

    2010-06-29

    The first step in assembly of membrane and storage glycerolipids is acylation of glycerol-3-phosphate (G3P). All previously characterized membrane-bound, eukaryotic G3P acyltransferases (GPATs) acylate the sn-1 position to produce lysophosphatidic acid (1-acyl-LPA). Cutin is a glycerolipid with omega-oxidized fatty acids and glycerol as integral components. It occurs as an extracellular polyester on the aerial surface of all plants, provides a barrier to pathogens and resistance to stress, and maintains organ identity. We have determined that Arabidopsis acyltransferases GPAT4 and GPAT6 required for cutin biosynthesis esterify acyl groups predominantly to the sn-2 position of G3P. In addition, these acyltransferases possess a phosphatase domain that results in sn-2 monoacylglycerol (2-MAG) rather than LPA as the major product. Such bifunctional activity has not been previously described in any organism. The possible roles of 2-MAGs as intermediates in cutin synthesis are discussed. GPAT5, which is essential for the accumulation of suberin aliphatics, also exhibits a strong preference for sn-2 acylation. However, phosphatase activity is absent and 2-acyl-LPA is the major product. Clearly, plant GPATs can catalyze more reactions than the sn-1 acylation by which they are currently categorized. Close homologs of GPAT4-6 are present in all land plants, but not in animals, fungi or microorganisms (including algae). Thus, these distinctive acyltransferases may have been important for evolution of extracellular glycerolipid polymers and adaptation of plants to a terrestrial environment. These results provide insight into the biosynthetic assembly of cutin and suberin, the two most abundant glycerolipid polymers in nature. PMID:20551224

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

    PubMed

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

    2016-01-01

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

  18. A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats.

    PubMed

    Mugford, Sam T; Qi, Xiaoquan; Bakht, Saleha; Hill, Lionel; Wegel, Eva; Hughes, Richard K; Papadopoulou, Kalliopi; Melton, Rachel; Philo, Mark; Sainsbury, Frank; Lomonossoff, George P; Roy, Abhijeet Deb; Goss, Rebecca J M; Osbourn, Anne

    2009-08-01

    Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense. PMID:19684243

  19. Alcoholism (image)

    MedlinePlus

    ... that interferes with physical or mental health, and social, family or job responsibilities. This addiction can lead to liver, circulatory and neurological problems. Pregnant women who drink alcohol in any amount ...

  20. Alcohol withdrawal

    MedlinePlus

    ... Seeing or feeling things that aren't there (hallucinations) Seizures Severe confusion ... alcohol withdrawal. You will be watched closely for hallucinations and other signs of delirium tremens. Treatment may ...

  1. Alcohol Abuse: Alcohol Withdrawal Syndrome

    MedlinePlus

    ... they quit drinking. What are the symptoms of alcohol withdrawal syndrome? Symptoms can be mild or severe, and may include: Shakiness Sweats Anxiety Irritability Fatigue Depression Headaches Insomnia Nightmares Decreased appetite More severe withdrawal symptoms ...

  2. Alcohol withdrawal.

    PubMed

    Manasco, Anton; Chang, Shannon; Larriviere, Joseph; Hamm, L Lee; Glass, Marcia

    2012-11-01

    Alcohol withdrawal is a common clinical condition that has a variety of complications and morbidities. The manifestations can range from mild agitation to withdrawal seizures and delirium tremens. The treatments for alcohol withdrawal include benzodiazepines, anticonvulsants, beta-blockers and antihypertensives. Although benzodiazepines are presently a first-line therapy, there is controversy regarding the efficacies of these medications compared with others. Treatment protocols often involve one of two contrasting approaches: symptom-triggered versus fixed-schedule dosing of benzodiazepines. We describe these protocols in our review and examine the data supporting symptom-triggered dosing as the preferred method for most patients in withdrawal.The Clinical Institute Withdrawal Assessment for Alcohol scoring system for alcohol withdrawal streamlines care, optimizes patient management, and is the best scale available for withdrawal assessment. Quality improvement implications for inpatient management of alcohol withdrawal include increasing training for signs of withdrawal and symptom recognition, adding new hospital protocols to employee curricula, and ensuring manageable patient-to-physician and patient-to-nurse ratios. PMID:23128805

  3. Structural and Functional Studies of a trans-Acyltransferase Polyketide Assembly Line Enzyme that Catalyzes Stereoselective α- and β-Ketoreduction

    PubMed Central

    Piasecki, Shawn K.; Zheng, Jianting; Axelrod, Abram J.; Detelich, Madeline; Keatinge-Clay, Adrian T.

    2014-01-01

    While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This ketoreductase naturally reduces both α- and β-keto groups and is the only ketoreductase known to do so during the biosynthesis of a polyketide. The isolated ketoreductase not only reduced an N-acetylcysteamine-bound β-keto substrate to a D-β-hydroxy product, but also an N-acetylcysteamine- bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a β-ketoreductase from a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase ketoreductases reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase ketoreductases, is predictive of the orientation of the resulting β-hydroxyl group. PMID:24634061

  4. Poly specific trans-acyltransferase machinery revealed via engineered acyl-CoA synthetases.

    PubMed

    Koryakina, Irina; McArthur, John; Randall, Shan; Draelos, Matthew M; Musiol, Ewa M; Muddiman, David C; Weber, Tilmann; Williams, Gavin J

    2013-01-18

    Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides. PMID:23083014

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

    PubMed Central

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

    2015-01-01

    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. PMID:26334609

  6. Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice

    PubMed Central

    Manzini, S.; Pinna, C.; Busnelli, M.; Cinquanta, P.; Rigamonti, E.; Ganzetti, G.S.; Dellera, F.; Sala, A.; Calabresi, L.; Franceschini, G.; Parolini, C.; Chiesa, G.

    2015-01-01

    Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcatwt) and LCAT knockout (LcatKO) mice exposed to noradrenaline showed reduced contractility in LcatKO mice (P < 0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in LcatKO mice (P < 0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in LcatKO mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcatwt and LcatKO mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

  7. Beta2-adrenergic activity modulates vascular tone regulation in lecithin:cholesterol acyltransferase knockout mice.

    PubMed

    Manzini, S; Pinna, C; Busnelli, M; Cinquanta, P; Rigamonti, E; Ganzetti, G S; Dellera, F; Sala, A; Calabresi, L; Franceschini, G; Parolini, C; Chiesa, G

    2015-11-01

    Lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with hypoalphalipoproteinemia, generally a predisposing factor for premature coronary heart disease. The evidence of accelerated atherosclerosis in LCAT-deficient subjects is however controversial. In this study, the effect of LCAT deficiency on vascular tone and endothelial function was investigated in LCAT knockout mice, which reproduce the human lipoprotein phenotype. Aortas from wild-type (Lcat(wt)) and LCAT knockout (Lcat(KO)) mice exposed to noradrenaline showed reduced contractility in Lcat(KO) mice (P<0.005), whereas acetylcholine exposure showed a lower NO-dependent relaxation in Lcat(KO) mice (P<0.05). Quantitative PCR and Western blotting analyses suggested an adequate eNOS expression in Lcat(KO) mouse aortas. Real-time PCR analysis indicated increased expression of β2-adrenergic receptors vs wild-type mice. Aorta stimulation with noradrenaline in the presence of propranolol, to abolish the β-mediated relaxation, showed the same contractile response in the two mouse lines. Furthermore, propranolol pretreatment of mouse aortas exposed to L-NAME prevented the difference in responses between Lcat(wt) and Lcat(KO) mice. The results indicate that LCAT deficiency leads to increased β2-adrenergic relaxation and to a consequently decreased NO-mediated vasodilation that can be reversed to guarantee a correct vascular tone. The present study suggests that LCAT deficiency is not associated with an impaired vascular reactivity. PMID:26254103

  8. Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

    NASA Technical Reports Server (NTRS)

    Malak, H.; Mahtani, H.; Herman, P.; Vecer, J.; Lu, X.; Chang, T. Y.; Richmond, Robert C.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

  9. Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

    PubMed Central

    Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

    2013-01-01

    Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

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

    PubMed

    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. PMID:27164260

  11. Expression of Fungal diacylglycerol acyltransferase2 Genes to Increase Kernel Oil in Maize[OA

    PubMed Central

    Oakes, Janette; Brackenridge, Doug; Colletti, Ron; Daley, Maureen; Hawkins, Deborah J.; Xiong, Hui; Mai, Jennifer; Screen, Steve E.; Val, Dale; Lardizabal, Kathryn; Gruys, Ken; Deikman, Jill

    2011-01-01

    Maize (Zea mays) oil has high value but is only about 4% of the grain by weight. To increase kernel oil content, fungal diacylglycerol acyltransferase2 (DGAT2) genes from Umbelopsis (formerly Mortierella) ramanniana and Neurospora crassa were introduced into maize using an embryo-enhanced promoter. The protein encoded by the N. crassa gene was longer than that of U. ramanniana. It included 353 amino acids that aligned to the U. ramanniana DGAT2A protein and a 243-amino acid sequence at the amino terminus that was unique to the N. crassa DGAT2 protein. Two forms of N. crassa DGAT2 were tested: the predicted full-length protein (L-NcDGAT2) and a shorter form (S-NcDGAT2) that encoded just the sequences that share homology with the U. ramanniana protein. Expression of all three transgenes in maize resulted in small but statistically significant increases in kernel oil. S-NcDGAT2 had the biggest impact on kernel oil, with a 26% (relative) increase in oil in kernels of the best events (inbred). Increases in kernel oil were also obtained in both conventional and high-oil hybrids, and grain yield was not affected by expression of these fungal DGAT2 transgenes. PMID:21245192

  12. Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase

    SciTech Connect

    Fielding, P.E.; Miida, Takashi; Fielding, C.J. )

    1991-09-03

    The metabolism of cholesterol derived from ({sup 3}H) cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large {alpha}-migrating HDL (HDL{sub 2}) and was then transferred to small {alpha}-HDL (HDL{sub 3}) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating HDL{sub 2}. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL{sub 2} as a relatively unreactive sink for LCAT-derived cholesteryl esters.

  13. Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum.

    PubMed

    Chen, Zhihuan; Liu, Pengyan; Liu, Yanhua; Tang, Hui; Chen, Yunping; Zhang, Liping

    2014-12-01

    Triacylglycerols (TAGs), synthesized in the microsomal membranes of eukaryotes, serve as a primary storage form of carbon and energy in microorganisms. For this reason, TAGs produced by organisms have great potential to become biofuels and facilitate researchers to look for alternative renewable sources of energy. The present study describes the identification and functional characterization of a type-2 diacylglycerol acyltransferase from Rhodosporidium diobovatum, designated as RdDGAT, which catalyzed the final step of TAG synthesis. A full-length cDNA clone for RdDGAT was obtained, and its biological activity was proven by being expressed in a Saccharomyces cerevisiae quadruple mutant that was defective in TAG synthesis. Enzymatic assays were performed and finally the existence of TAGs in the transformed Saccharomyces cerevisiae quadruple mutant was determined using the method of thin-layer chromatography. Substrate preference experiments revealed that RdDGAT preferred unsaturated fatty acids over saturated ones. Through further analysis, we assume that the evolution and expression characteristics of the RdDGAT gene perhaps is the result of adaption to its oligotrophic and cold living environment. PMID:25294723

  14. Abrogating monoacylglycerol acyltransferase activity in liver improves glucose tolerance and hepatic insulin signaling in obese mice.

    PubMed

    Hall, Angela M; Soufi, Nisreen; Chambers, Kari T; Chen, Zhouji; Schweitzer, George G; McCommis, Kyle S; Erion, Derek M; Graham, Mark J; Su, Xiong; Finck, Brian N

    2014-07-01

    Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol (DAG), a lipid that has been linked to the development of hepatic insulin resistance through activation of protein kinase C (PKC). The expression of genes that encode MGAT enzymes is induced in the livers of insulin-resistant human subjects with nonalcoholic fatty liver disease, but whether MGAT activation is causal of hepatic steatosis or insulin resistance is unknown. We show that the expression of Mogat1, which encodes MGAT1, and MGAT activity are also increased in diet-induced obese (DIO) and ob/obmice. To probe the metabolic effects of MGAT1 in the livers of obese mice, we administered antisense oligonucleotides (ASOs) against Mogat1 to DIO and ob/ob mice for 3 weeks. Knockdown of Mogat1 in liver, which reduced hepatic MGAT activity, did not affect hepatic triacylglycerol content and unexpectedly increased total DAG content. Mogat1 inhibition also increased both membrane and cytosolic compartment DAG levels. However, Mogat1 ASO treatment significantly improved glucose tolerance and hepatic insulin signaling in obese mice. In summary, inactivation of hepatic MGAT activity, which is markedly increased in obese mice, improved glucose tolerance and hepatic insulin signaling independent of changes in body weight, intrahepatic DAG and TAG content, and PKC signaling. PMID:24595352

  15. Cloning and Functional Analysis of Three Diacylglycerol Acyltransferase Genes from Peanut (Arachis hypogaea L.)

    PubMed Central

    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. PMID:25181516

  16. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

    PubMed Central

    Ye, Zhixia; Musiol, Ewa M; Weber, Tilmann; Williams, Gavin J

    2014-01-01

    SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues. PMID:24726832

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

    PubMed

    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. PMID:26454063

  18. Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity

    PubMed Central

    McMahon, Derek; Dinh, Anna; Kurz, Daniel; Shah, Dharika; Han, Gil-Soo; Carman, George M.; Brasaemle, Dawn L.

    2014-01-01

    Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity. PMID:24879803

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

    PubMed

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

    2015-01-01

    The esterification of methylecgonine (2-carbomethoxy-3β-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots. PMID:25406120

  20. The Last Step in Cocaine Biosynthesis Is Catalyzed by a BAHD Acyltransferase[OPEN

    PubMed Central

    Schmidt, Gregor Wolfgang; Porta, Tiffany; Reichelt, Michael; Luck, Katrin; Torre, José Carlos Pardo; Dolke, Franziska; Varesio, Emmanuel; Hopfgartner, Gérard; Gershenzon, Jonathan

    2015-01-01

    The esterification of methylecgonine (2-carbomethoxy-3β-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots. PMID:25406120

  1. Molecular and phylogenetic analysis of pyridoxal phosphate-dependent acyltransferase of Exiguobacterium acetylicum.

    PubMed

    Rajendran, Narayanan; Smith, Colby; Mazhawidza, Williard

    2009-01-01

    The pyridoxal-5'-phosphate (PLP)-dependent family of enzymes is a very diverse group of proteins that metabolize small molecules like amino acids and sugars, and synthesize cofactors for other metabolic pathways through transamination, decarboxylation, racemization, and substitution reactions. In this study we employed degenerated primer-based PCR amplification, using genomic DNA isolated from the soil bacterium Exiguobacterium acetylicum strain SN as template. We revealed the presence of a PLP-dependent family of enzymes, such as PLP-dependent acyltransferase, and similarity to 8-amino-7-oxononoate synthase. Sequencing analysis and multiple alignment of the thymidine-adenine-cloned PCR amplicon revealed PLP-dependent family enzymes with specific confering codes and consensus amino acid residues specific to this group of functional proteins. Amino acid residues common to the majority of PLP-dependent enzymes were also revealed by the Lasergene MegAlign software. A phylogenetic tree was constructed. Its analysis revealed a close relationship of E. acetylicum to other bacteria isolated from extreme environments suggesting similarities in anabolic adaptability and evolutionary development. PMID:20158163

  2. Phosphatidic acid phosphatase and diacylglycerol acyltransferase: potential targets for metabolic engineering of microorganism oil.

    PubMed

    Jin, Hong-Hao; Jiang, Jian-Guo

    2015-04-01

    Oleaginous microorganism is becoming one of the most promising oil feedstocks for biodiesel production due to its great advantages in triglyceride (TAG) accumulation. Previous studies have shown that de novo TAG biosynthesis can be divided into two parts: the fatty acid biosynthesis pathway (the upstream part which generates acyl-CoAs) and the glycerol-3-phosphate acylation pathway (the downstream part in which three acyl groups are sequentially added onto a glycerol backbone). This review mainly focuses on two enzymes in the G3P pathway, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). The former catalyzes a dephosphorylation reaction, and the latter catalyzes a subsequent acylation reaction. Genes, functional motifs, transmembrane domains, action mechanism, and new studies of the two enzymes are discussed in detail. Furthermore, this review also covers diacylglycerol kinase, an enzyme that catalyzes the reverse reaction of diacylglycerol formation. In addition, PAP and DGAT are the conjunction points of the G3P pathway, the Kennedy pathway, and the CDP-diacylglycerol pathway (CDP-DAG pathway), and the mutual transformation between TAGs and phospholipids is discussed as well. Given that both the Kennedy and CDP-diacylglycerol pathways are in metabolic interlock (MI) with the G3P pathway, it is suggested that, via metabolic engineering, TAG accumulation can be improved by the two pathways based on the pivotal function of PAP and DGAT. PMID:25672855

  3. Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase.

    PubMed

    Greer, Michael S; Truksa, Martin; Deng, Wei; Lung, Shiu-Cheung; Chen, Guanqun; Weselake, Randall J

    2015-03-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs' native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications. PMID:25520169

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

    PubMed

    Netherland, Courtney; Thewke, Douglas P

    2010-08-01

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

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

    PubMed

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

    1996-04-01

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

  6. Selectivity of microbial acyl-CoA: cholesterol acyltransferase inhibitors toward isozymes.

    PubMed

    Ohshiro, Taichi; Rudel, Lawrence L; Omura, Satoshi; Tomoda, Hiroshi

    2007-01-01

    The selectivity of microbial inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT) toward the two isozymes, ACAT1 and ACAT2, was assessed in cell-based assays. Purpactin A (IC50 values of ACAT1 vs. IC50 values of ACAT2; 2.5 microM vs. 1.5 microM), terpendole C (10 microM vs. 10 microM), glisoprenin A (4.3 microM vs. 10 microM), spylidone (25 microM vs. 5.0 microM) and synthetic CL-283,546 (0.1 microM vs. 0.09 microM) inhibited ACAT1 and ACAT2 to similar extents. Beauveriolides I (0.6 microM vs. 20 microM) and III (0.9 microM vs. >20 microM) inhibited ACAT1 rather selectively, while pyripyropenes A (>80 microM vs. 0.07 microM), B (48 microM vs. 2.0 microM), C (32 microM vs. 0.36 microM) and D (38 microM vs. 1.5 microM) showed selective inhibition against ACAT2. In particular, pyripyropene A was found to be the most selective ACAT2 inhibitor with a selective index of more than 1,000. PMID:17390588

  7. Acute Sterol O-Acyltransferase 2 (SOAT2) Knockdown Rapidly Mobilizes Hepatic Cholesterol for Fecal Excretion

    PubMed Central

    Marshall, Stephanie M.; Gromovsky, Anthony D.; Kelley, Kathryn L.; Davis, Matthew A.; Wilson, Martha D.; Lee, Richard G.; Crooke, Rosanne M.; Graham, Mark J.; Rudel, Lawrence L.

    2014-01-01

    The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion. PMID:24901470

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

    PubMed Central

    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

  9. Fetal Alcohol Spectrum Disorders

    MedlinePlus

    ... alcohol can cause a group of conditions called fetal alcohol spectrum disorders (FASDs). Effects can include physical and behavioral problems such ... alcohol syndrome is the most serious type of FASD. People with fetal alcohol syndrome have facial abnormalities, ...

  10. Naltrexone for Alcoholism

    MedlinePlus

    MENU Return to Web version Naltrexone for Alcoholism Naltrexone for Alcoholism Is alcoholism a disease? Yes. Most experts agree that alcoholism is a disease, just as high blood pressure, diabetes and ...

  11. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

    DOE PAGESBeta

    Poust, Sean; Yoon, Isu; Adams, Paul D.; Katz, Leonard; Petzold, Christopher J.; Keasling, Jay D.

    2014-10-06

    Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-likemore » subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.« less

  12. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

    SciTech Connect

    Poust, Sean; Yoon, Isu; Adams, Paul D.; Katz, Leonard; Petzold, Christopher J.; Keasling, Jay D.

    2014-10-06

    Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.

  13. A Double-Hotdog with a New Trick: Structure and Mechanism of the trans-Acyltransferase Polyketide Synthase Enoyl-isomerase

    PubMed Central

    2015-01-01

    Many polyketide natural products exhibit invaluable medicinal properties, yet much remains to be understood regarding the machinery responsible for their biosynthesis. The recently discovered trans-acyltransferase polyketide synthases employ processing enzymes that catalyze modifications unique from those of the classical cis-acyltransferase polyketide synthases. The enoyl-isomerase domains of these megasynthases shift double bonds and are well-represented by an enzyme that helps forge the triene system within the antibiotic produced by the prototypical bacillaene synthase. This first crystal structure of an enoyl-isomerase, at 1.73 Å resolution, not only revealed relationships between this class of enzymes and dehydratases but also guided an investigation into the mechanism of double bond migration. The catalytic histidine, positioned differently from that of dehydratases, was demonstrated to independently shuttle a proton between the γ- and α-positions of the intermediate. This unprecedented mechanism highlights the catalytic diversity of divergent enzymes within trans-acyltransferase polyketide synthases. PMID:25089587

  14. Alcohol fuels

    SciTech Connect

    Not Available

    1981-07-01

    The API publication 4312 reports a detailed study carried out by Battelle on the energy balances for five alcohol-fuel-producing technologies. The results indicate that processes for producing ethanol from corn are net consumers of energy while ethanol from sugar cane and methanol from wood are net energy producers.

  15. Allyl alcohol

    Integrated Risk Information System (IRIS)

    Allyl alcohol ; CASRN 107 - 18 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Eff

  16. Isobutyl alcohol

    Integrated Risk Information System (IRIS)

    Isobutyl alcohol ; CASRN 78 - 83 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E

  17. Propargyl alcohol

    Integrated Risk Information System (IRIS)

    Propargyl alcohol ; CASRN 107 - 19 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  18. Alcoholism and Minority Populations.

    ERIC Educational Resources Information Center

    Watts, Thomas D.; Wright, Roosevelt, Jr.

    1991-01-01

    Briefly discusses some aspects of the role of the state and the position of minorities in respect to alcoholism policies and services. Includes case study of a Black alcoholic. Refers readers to studies on Black alcoholism, Native American alcoholism, Hispanic alcoholism, and Asian-American alcoholism. (Author/NB)

  19. Novel serum biomarkers for detection of excessive alcohol use

    PubMed Central

    Liangpunsakul, Suthat; Lai, Xianyin; Ross, Ruth A.; Yu, Zhangsheng; Modlik, Elizabeth; Westerhold, Chi; Heathers, Laura; Paul, Robin; O’Connor, Sean; Crabb, David W.; Witzmann, Frank

    2015-01-01

    Objectives Construct interview that correctly identifies those with alcohol use disorder have limitation, especially when the subjects are motivated to minimize the magnitude of drinking behavior. Current laboratory tests to detect excessive alcohol consumption are limited by marginal sensitivity/specificity. Excessive drinking has been shown to affect several organ systems; which may be reflected in changes in quantity of plasma proteins. Our aim was to employ novel proteomic analyses to identify potential markers for excessive alcohol use. METHODS A prospective case-control study that included 39 controls and 54 excessive drinkers (discovery cohort). The serum proteomic analyses in these subjects were performed and the results were tested in the verification cohort (40 controls and 40 excessive drinkers). RESULTS Using the appropriate cutoff and confirmation with ELISA, we identified 4 proteins which were significantly elevated in the serum of excessive drinkers; AT-rich interactive domain-containing protein 4B (ARID4B), Phosphatidylcholine-sterol acyltransferase (LCAT), Hepatocyte growth factor-like protein (MST1), and ADP-ribosylation factor 6 (ARL6). The performance of the conventional markers (AST, ALT, GGT, %CDT, and MCV) discriminating between excessive alcohol use and controls had an area under the curve (AUC) ranging from 0.21 (ALT) to 0.67 (MCV). The AUC of these novel proteins showed the improvement in the detection of excessive drinkers compared to conventional lab tests, ranging from 0.73 (for ARID4B) to 0.86 (for ARL6). CONCLUSIONS We have identified four novel proteins that can discern subjects with excessive alcohol use. Further studies are needed to determine the clinical implications of these markers to detect excessive alcohol use and confirm abstinence. PMID:25704570

  20. Alcohol exposure in utero perturbs retinoid homeostasis in adult rats

    PubMed Central

    Kim, Youn-Kyung; Zuccaro, Michael V.; Zhang, Changqing; Sarkar, Dipak

    2015-01-01

    Background Maternal alcohol exposure and adult alcohol intake have been shown to perturb the metabolism of various micro- and macro-nutrients, including vitamin A and its derivatives (retinoids). Therefore, it has been hypothesized that the well-known detrimental consequences of alcohol consumption may be due to deregulations of the metabolism of such nutrients rather than to a direct effect of alcohol. Alcohol exposure in utero also has long-term harmful consequences on the health of the offspring with mechanisms that have not been fully clarified. Disruption of tissue retinoid homeostasis has been linked not only to abnormal embryonic development, but also to various adult pathological conditions, including cancer, metabolic disorders and abnormal lung function. We hypothesized that prenatal alcohol exposure may permanently perturb tissue retinoid metabolism, predisposing the offspring to adult chronic diseases. Methods Serum and tissues (liver, lung and prostate from males; liver and lung from females) were collected from 60-75 day-old sprague dawley rats born from dams that were: (I) fed a liquid diet containing 6.7% alcohol between gestational day 7 and 21; or (II) pair-fed with isocaloric liquid diet during the same gestational window; or (III) fed ad libitum with regular rat chow diet throughout pregnancy. Serum and tissue retinoid levels were analyzed by reverse-phase high-performance liquid chromatography (HPLC). Serum retinol-binding protein (RBP) levels were measured by western blot analysis, and liver, lung and prostate mRNA levels of lecithin-retinol acyltransferase (LRAT) were measured by qPCR. Results Retinyl ester levels were significantly reduced in the lung of both males and females, as well as in the liver and ventral prostate of males born from alcohol-fed dams. Tissue LRAT mRNA levels remained unchanged upon maternal alcohol treatment. Conclusions Prenatal alcohol exposure in rats affects retinoid metabolism in adult life, in a tissue- and sex

  1. Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

    PubMed

    Van, Bo; Nishi, Miyuki; Komazaki, Shinji; Ichimura, Atsuhiko; Kakizawa, Sho; Nakanaga, Keita; Aoki, Junken; Park, Ki-Ho; Ma, Jianjie; Ueyama, Tomomi; Ogata, Takehiro; Maruyama, Naoki; Takeshima, Hiroshi

    2015-04-28

    Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation. PMID:25841338

  2. Severe high-density lipoprotein deficiency associated with autoantibodies against lecithin:cholesterol acyltransferase in non-Hodgkin lymphoma.

    PubMed

    Simonelli, Sara; Gianazza, Elisabetta; Mombelli, Giuliana; Bondioli, Alighiero; Ferraro, Giovanni; Penco, Silvana; Sirtori, Cesare R; Franceschini, Guido; Calabresi, Laura

    2012-01-23

    An antibody against the lecithin:cholesterol acyltransferase (LCAT) enzyme, which negates cholesterol esterification in plasma, causing severe high-density lipoprotein deficiency (HD), was identified in a woman with a large-cell non-Hodgkin lymphoma. Successful treatment of the lymphoma resulted in clearance of the antibody and complete correction of the defective cholesterol esterification and HD. To our knowledge, an acquired LCAT deficiency leading to severe HD has not been reported previously in association with a malignant disease, and this patient represents the first such documented case. PMID:22271127

  3. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.

    PubMed

    Tomoda, H; Kim, Y K; Nishida, H; Masuma, R; Omura, S

    1994-02-01

    Aspergillus fumigatus FO-1289, a soil isolate, was found to produce a series of novel inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Four active compounds, named pyripyropenes A, B, C and D, were isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. Pyripyropenes A, B, C and D show very potent ACAT inhibitory activity in an enzyme assay system using rat liver microsomes with IC50 values of 58, 117, 53 and 268 nM, respectively. PMID:8150709

  4. Ldl modified by hypochlorous acid is a potent inhibitor of lecithin-cholesterol acyltransferase activity.

    PubMed

    McCall, M R; Carr, A C; Forte, T M; Frei, B

    2001-06-01

    Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily N-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 micromol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B-depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in N-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 micromol/L HOCl, respectively. Addition of 200 micromol/L ascorbate or N-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with

  5. Crystal structure of the acyltransferase domain of the iterative polyketide synthase in enediyne biosynthesis.

    PubMed

    Liew, Chong Wai; Nilsson, Martina; Chen, Ming Wei; Sun, Huihua; Cornvik, Tobias; Liang, Zhao-Xun; Lescar, Julien

    2012-06-29

    Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT(DYN10)) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a α/β hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a α/β fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude α-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser(651) residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates. PMID:22589546

  6. Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

    PubMed Central

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

    2013-01-01

    Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms. PMID:24146944

  7. A Lipolytic Lecithin:Cholesterol Acyltransferase Secreted by Toxoplasma Facilitates Parasite Replication and Egress.

    PubMed

    Pszenny, Viviana; Ehrenman, Karen; Romano, Julia D; Kennard, Andrea; Schultz, Aric; Roos, David S; Grigg, Michael E; Carruthers, Vern B; Coppens, Isabelle

    2016-02-19

    The protozoan parasite Toxoplasma gondii develops within a parasitophorous vacuole (PV) in mammalian cells, where it scavenges cholesterol. When cholesterol is present in excess in its environment, the parasite expulses this lipid into the PV or esterifies it for storage in lipid bodies. Here, we characterized a unique T. gondii homologue of mammalian lecithin:cholesterol acyltransferase (LCAT), a key enzyme that produces cholesteryl esters via transfer of acyl groups from phospholipids to the 3-OH of free cholesterol, leading to the removal of excess cholesterol from tissues. TgLCAT contains a motif characteristic of serine lipases "AHSLG" and the catalytic triad consisting of serine, aspartate, and histidine (SDH) from LCAT enzymes. TgLCAT is secreted by the parasite, but unlike other LCAT enzymes it is cleaved into two proteolytic fragments that share the residues of the catalytic triad and need to be reassembled to reconstitute enzymatic activity. TgLCAT uses phosphatidylcholine as substrate to form lysophosphatidylcholine that has the potential to disrupt membranes. The released fatty acid is transferred to cholesterol, but with a lower transesterification activity than mammalian LCAT. TgLCAT is stored in a subpopulation of dense granule secretory organelles, and following secretion, it localizes to the PV and parasite plasma membrane. LCAT-null parasites have impaired growth in vitro, reduced virulence in animals, and exhibit delays in egress from host cells. Parasites overexpressing LCAT show increased virulence and faster egress. These observations demonstrate that TgLCAT influences the outcome of an infection, presumably by facilitating replication and egress depending on the developmental stage of the parasite. PMID:26694607

  8. Identification of genetic variants of lecithin cholesterol acyltransferase in individuals with high HDL‑C levels.

    PubMed

    Naseri, Mohsen; Hedayati, Mehdi; Daneshpour, Maryam Sadat; Bandarian, Fatemeh; Azizi, Fereidoun

    2014-07-01

    Among the most common lipid abnormalities, a low level of high-density lipoprotein-cholesterol (HDL‑C) is one of the first risk factors identified for coronary heart disease. Lecithin cholesterol acyltransferase (LCAT) has a pivotal role in the formation and maturation of HDL-C and in reverse cholesterol transport. To identify genetic loci associated with low HDL-C in a population-based cohort in Tehran, the promoter, coding regions and exon/intron boundaries of LCAT were amplified and sequenced in consecutive individuals (n=150) who had extremely low or high HDL-C levels but no other major lipid abnormalities. A total of 14 single-nucleotide polymorphisms (SNPs) were identified, of which 10 were found to be novel; the L393L, S232T and 16:67977696 C>A polymorphisms have been previously reported in the SNP Database (as rs5923, rs4986970 and rs11860115, respectively) and the non-synonymous R47M mutation has been reported in the Catalogue of Somatic Mutations in Cancer (COSM972635). Three of the SNPs identified in the present study (position 6,531 in exon 5, position 6,696 in exon 5 and position 5,151 in exon 1) led to an amino acid substitution. The most common variants were L393L (4886C/T) in exon 6 and Q177E, a novel mutation, in exon 5, and the prevalence of the heterozygous genotype of these two SNPs was significantly higher in the low HDL-C groups. Univariate conditional logistic regression odds ratios (ORs) were nominally significant for Q177E (OR, 5.64; P=0.02; 95% confidence interval, 1.2‑26.2). However, this finding was attenuated following adjustment for confounders. Further studies using a larger sample size may enhance the determination of the role of these SNPs. PMID:24789697

  9. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism.

    PubMed

    Gunawardane, Ruwanthi N; Fordstrom, Preston; Piper, Derek E; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

    2016-02-01

    Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouse(TM) platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

  10. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol.

    PubMed

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G; Browse, John

    2015-10-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop. PMID:26195728

  11. Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

    PubMed Central

    Rastegar, Farbod; Gao, Jian-Li; Shenaq, Deana; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Jiang, Wei; Wagner, Eric R.; Huang, Enyi; Gao, Yanhong; Shen, Jikun; Yang, Ke; He, Bai-Cheng; Chen, Liang; Zuo, Guo-Wei; Luo, Jinyong; Luo, Xiaoji; Bi, Yang; Liu, Xing; Li, Mi; Hu, Ning; Wang, Linyuan; Luther, Gaurav; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan

    2010-01-01

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

  12. Expression, Purification, and Characterization of Mouse Glycine N-acyltransferase in Escherichia coli

    PubMed Central

    Dempsey, Daniel R.; Bond, Jason D.; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J.

    2014-01-01

    Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34 kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5 mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app = (5.2 ± 0.20) × 102M−1s−1, and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app = (4.5 ± 0.27) × 105M−1s−1. Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2-C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids. PMID:24576660

  13. Inhibiting monoacylglycerol acyltransferase 1 ameliorates hepatic metabolic abnormalities but not inflammation and injury in mice.

    PubMed

    Soufi, Nisreen; Hall, Angela M; Chen, Zhouji; Yoshino, Jun; Collier, Sara L; Mathews, James C; Brunt, Elizabeth M; Albert, Carolyn J; Graham, Mark J; Ford, David A; Finck, Brian N

    2014-10-24

    Abnormalities in hepatic lipid metabolism and insulin action are believed to play a critical role in the etiology of nonalcoholic steatohepatitis. Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis, and knocking down Mogat1 improves glucose metabolism and hepatic insulin signaling, but whether increased MGAT activity plays a role in the etiology of nonalcoholic steatohepatitis is unclear. To examine this issue, mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were injected with antisense oligonucleotides (ASOs) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. The HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver and adipose tissue, attenuated weight gain, improved glucose tolerance, improved hepatic insulin signaling, and decreased hepatic triacylglycerol content compared with control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic diacylglycerol, cholesterol, or free fatty acid content; improve histologic measures of liver injury; or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves hepatic metabolic abnormalities without attenuating liver inflammation and injury. PMID:25213859

  14. Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

    PubMed Central

    Hoeg, J M; Santamarina-Fojo, S; Bérard, A M; Cornhill, J F; Herderick, E E; Feldman, S H; Haudenschild, C C; Vaisman, B L; Hoyt, R F; Demosky, S J; Kauffman, R D; Hazel, C M; Marcovina, S M; Brewer, H B

    1996-01-01

    Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis. Images Fig. 2 Fig. 3 Fig. 4 PMID:8876155

  15. Regulation of plasma lecithin:cholesterol acyltransferase. II. Activation during alimentary lipemia.

    PubMed

    Rose, H G; Juliano, J

    1977-03-01

    The effect of dietary fat on plasma lecithin:cholesterol acyltransferase (LCAT) activity has been investigated in 14 normal male subjects. After determination of postabsorptive lipid and LCAT levels, a high-fat liquid test meal (1 to 2 gm./kg. body weight) was fed, followed by lipid and LCAT determinations at 2.5 hour intervals. Plasma triglycerides were elevated by 2.5 hours, peaked at 5.0 hours, fell at 7.5 hours, and were normalized by 10 hours. LCAT was unchanged at 2.5 hours but was elevated by 5.0 hours, exhibiting a broad plateau through 10 hours. Most subjects manifested peak responses at 7.5 hours. The mean maximal increase in individual subjects was 37.2 +/- 13.3 (S.D.) percent. LCAT changes similarly followed the elevation and recession of chylomicrons (Sf greater than 400) and very-low-density lipoprotein triglycerides, both of which closely paralleled plasma triglycerides. Enzyme responses were proportional to percentage elevations of plasma triglycerides (r = 0.93, p less than 0.01) and related to quantity of fat in the test diet. Three subjects who ingested the test diet devoid of the fat component showed no significant change in enzyme activity. Enzyme progress curves revealed linearity for 3 hours for both postabsorptive and lipemic (7.5 hour) plasma from the same subjects, supporting the validity of the assay as a measure of enzyme rate. These studies demonstrate an increase in cholesterol esterifying activity temporally related to the clearance of alimentary particles, suggesting a physiologic role in the clearance process. PMID:839110

  16. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol

    PubMed Central

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G.; Browse, John

    2015-01-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world’s most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop. PMID:26195728

  17. Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism*

    PubMed Central

    Gunawardane, Ruwanthi N.; Fordstrom, Preston; Piper, Derek E.; Masterman, Stephanie; Siu, Sophia; Liu, Dongming; Brown, Mike; Lu, Mei; Tang, Jie; Zhang, Richard; Cheng, Janet; Gates, Andrew; Meininger, David; Chan, Joyce; Carlson, Tim; Walker, Nigel; Schwarz, Margrit; Delaney, John; Zhou, Mingyue

    2016-01-01

    Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease. PMID:26644477

  18. Inhibiting Monoacylglycerol Acyltransferase 1 Ameliorates Hepatic Metabolic Abnormalities but Not Inflammation and Injury in Mice*

    PubMed Central

    Soufi, Nisreen; Hall, Angela M.; Chen, Zhouji; Yoshino, Jun; Collier, Sara L.; Mathews, James C.; Brunt, Elizabeth M.; Albert, Carolyn J.; Graham, Mark J.; Ford, David A.; Finck, Brian N.

    2014-01-01

    Abnormalities in hepatic lipid metabolism and insulin action are believed to play a critical role in the etiology of nonalcoholic steatohepatitis. Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis, and knocking down Mogat1 improves glucose metabolism and hepatic insulin signaling, but whether increased MGAT activity plays a role in the etiology of nonalcoholic steatohepatitis is unclear. To examine this issue, mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were injected with antisense oligonucleotides (ASOs) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. The HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver and adipose tissue, attenuated weight gain, improved glucose tolerance, improved hepatic insulin signaling, and decreased hepatic triacylglycerol content compared with control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic diacylglycerol, cholesterol, or free fatty acid content; improve histologic measures of liver injury; or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves hepatic metabolic abnormalities without attenuating liver inflammation and injury. PMID:25213859

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

    PubMed

    Sears, Avery E; Palczewski, Krzysztof

    2016-06-01

    Lecithin:retinol acyltransferase (LRAT) catalyzes the acyl transfer from the sn-1 position of phosphatidylcholine (PC) to all-trans-retinol, creating fatty acid retinyl esters (palmitoyl, stearoyl, and some unsaturated derivatives). In the eye, these retinyl esters are substrates for the 65 kDa retinoid isomerase (RPE65). LRAT is well characterized biochemically, and recent structural data from closely related family members of the NlpC/P60 superfamily and a chimeric protein have established its catalytic mechanism. Mutations in the LRAT gene are responsible for approximately 1% of reported cases of Leber congenital amaurosis (LCA). Lack of functional LRAT, expressed in the retinal pigmented epithelium (RPE), results in loss of the visual chromophore and photoreceptor degeneration. LCA is a rare hereditary retinal dystrophy with an early onset associated with mutations in one of 21 known genes. Protocols have been devised to identify therapeutics that compensate for mutations in RPE65, also associated with LCA. The same protocols can be adapted to combat dystrophies associated with LRAT. Improvement in the visual function of clinical recipients of therapy with recombinant adeno-associated virus (rAAV) vectors incorporating the RPE65 gene provides a proof of concept for LRAT, which functions in the same cell type and metabolic pathway as RPE65. In parallel, a clinical trial that employs oral 9-cis-retinyl acetate to replace the missing chromophore in RPE65 and LRAT causative disease has proven to be effective and free of adverse effects. This article summarizes the biochemistry of LRAT and examines chromophore replacement as a treatment for LCA caused by LRAT mutations. PMID:27183166

  20. Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida Lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation.

    PubMed

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation. PMID:24204024

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

    PubMed

    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. PMID:23456478

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

    PubMed

    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. PMID:26645250

  3. Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice*

    PubMed Central

    Hager, Lauren; Li, Lixin; Pun, Henry; Liu, Lu; Hossain, Mohammad A.; Maguire, Graham F.; Naples, Mark; Baker, Chris; Magomedova, Lilia; Tam, Jonathan; Adeli, Khosrow; Cummins, Carolyn L.; Connelly, Philip W.; Ng, Dominic S.

    2012-01-01

    We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr−/−xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr−/−xLcat−/− mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr−/−xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr−/−xLcat−/− mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr−/−xLcat−/− mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr−/−xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr−/−xLcat−/− mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance. PMID:22500017

  4. Oral Carcinogenesis Induced by 4-Nitroquinoline 1-Oxide in Lecithin:retinol Acyltransferase Gene Knockout Mice

    PubMed Central

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

    2010-01-01

    Lecithin:retinol acyltransferase (LRAT) regulates retinol (vitamin A) metabolism by esterifying retinol. LRAT expression is decreased in cultured human head and neck squamous cell carcinoma (SCCHN) relative to normal epithelial cells. We investigated whether the carcinogen 4-nitroquinoline 1-oxide (4-NQO) induced a higher incidence of oral cancer in LRAT knockout than wild type (Wt) mice. We also investigated retinol deprivation during 4-NQO treatment in LRAT−/− mice as a model for rapid retinol deficiency. We observed higher levels of secreted frizzled-related protein 2 (Sfrp2), an inhibitor of WNT signaling, in tongue tumors in LRAT−/− versus Wt. LRAT−/− embryonic stem cells also expressed higher Sfrp2 transcripts, indicating an interaction between retinol and WNT signaling. Cox-2, Cyclin D1, p21, Trop2, and RARβ2 were not differentially expressed in Wt versus LRAT−/− tongue tumors. Wt and LRAT−/− mice fed a retinol sufficient diet showed the same oral tumor incidence after 4-NQO. In contrast, tongue tumors developed in 60% of Wt and in 100% of LRAT−/− mice fed a retinol deficient diet during 4-NQO treatment (p=0.22); moreover, the BrdU labeling index was 21.0±2.4% in LRAT−/− normal tongue epithelium as compared to 9.9±0.8% in Wt (p<0.001). Thus, partial retinol deficiency during carcinogen treatment (achieved in LRAT−/−) resulted in more proliferating cells in tongue epithelia from LRAT−/− mice and ultimately a greater probability of carcinogenesis. PMID:19954945

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

    PubMed

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

    2015-11-01

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

  6. Interstellar Alcohols

    NASA Technical Reports Server (NTRS)

    Charnley, S. B.; Kress, M. E.; Tielens, A. G. G. M.; Millar, T. J.

    1995-01-01

    We have investigated the gas-phase chemistry in dense cores where ice mantles containing ethanol and other alcohols have been evaporated. Model calculations show that methanol, ethanol, propanol, and butanol drive a chemistry leading to the formation of several large ethers and esters. Of these molecules, methyl ethyl ether (CH3OC2H5) and diethyl ether (C2H5)2O attain the highest abundances and should be present in detectable quantities within cores rich in ethanol and methanol. Gas-phase reactions act to destroy evaporated ethanol and a low observed abundance of gas-phase C,H,OH does not rule out a high solid-phase abundance. Grain surface formation mechanisms and other possible gas-phase reactions driven by alcohols are discussed, as are observing strategies for the detection of these large interstellar molecules.

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

    PubMed Central

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

    2005-01-01

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

  8. Formation of fatty acid ethyl esters in rat liver microsomes. Evidence for a key role for acyl-CoA: ethanol O-acyltransferase.

    PubMed

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

    1999-01-01

    Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non-oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl-CoA : ethanol O-acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol.min-1.mg-1). However, the AEAT activity was increased about sixfold by the addition of 250 microm bis-(4-nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p-Hydroxymercuribenzoic acid (a cysteine-reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl-CoA) and to a lesser extent the product by serine esterases. dl-Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 microm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES-4 and ES-10, and identified ES-4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES-4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl-CoA/non-esterified fatty acids is high under normal

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

    PubMed Central

    2010-01-01

    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 < 0.01 for all). The frequency of A and C alleles was 79.0% and 21.0% in Bai Ku Yao, and 87.3% and 12.7% in Han (P < 0.001); respectively. The frequency of AA, AC and CC genotypes was 63.2%, 31.4% and 5.2% in Bai Ku Yao, and 75.6%, 23.2% and 1.1% in Han (P < 0.001); respectively. The levels of TC, LDL-C and ApoB in Bai Ku Yao but not in Han were different between the AA and AC/CC genotypes in females but not in males (P < 0.05 for all). The C allele carriers had lower serum TC, LDL-C and ApoB levels as compared with the C allele noncarriers. The levels of TC, LDL-C and ApoB in Bai Ku Yao but not in Han were correlated with genotypes in females but not in males (P < 0.05 for all). Serum lipid parameters were also correlated with sex, age, body mass index, alcohol consumption, and blood pressure in both ethnic groups (P < 0.05-0.001). Conclusions These results suggest that the polymorphism of rs1044925 in the ACAT-1 gene is mainly associated with female serum TC, LDL-C and

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

    PubMed Central

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

    2016-01-01

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl–CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. PMID:26965057

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

    PubMed

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

    2016-01-01

    The biosynthesis of phospholipids and glycolipids are critical pathways for virtually all cell membranes. PatA is an essential membrane associated acyltransferase involved in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides (PIMs). The enzyme transfers a palmitoyl moiety from palmitoyl-CoA to the 6-position of the mannose ring linked to 2-position of inositol in PIM1/PIM2. We report here the crystal structures of PatA from Mycobacterium smegmatis in the presence of its naturally occurring acyl donor palmitate and a nonhydrolyzable palmitoyl-CoA analog. The structures reveal an α/β architecture, with the acyl chain deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. Our studies establish the mechanistic basis of substrate/membrane recognition and catalysis for an important family of acyltransferases, providing exciting possibilities for inhibitor design. PMID:26965057

  12. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making

    PubMed Central

    Tuladhar, Rubina; Lum, Lawrence

    2015-01-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  13. Fatty acyl donor selectivity in membrane bound O-acyltransferases and communal cell fate decision-making.

    PubMed

    Tuladhar, Rubina; Lum, Lawrence

    2015-04-01

    The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms. PMID:25849923

  14. Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues.

    PubMed

    Manaf, A M; Harwood, J L

    2000-01-01

    Glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.15) catalyses the first step of the Kennedy pathway for acyl lipid formation. This enzyme was studied using high-speed particulate fractions from oil palm (Elaeis guineensis Jacq.) tissue cultures and mesocarp acetone powders. The fractions were incubated with [(14)C]glycerol 3-phosphate and incorporation of radioactivity into Kennedy pathway intermediates studied. Optimal conditions were broadly similar between the two preparations but those from fruit mesocarp clearly contained more active enzymes for the subsequent stages of the Kennedy pathway - as exemplified by the appreciable accumulation of radioactivity in triacylglycerol. Experiments with different acyl-CoA substrates showed that the GPAT in both high-speed particulate preparations had a significant preference for palmitate. Glycerol 3-phosphate acyltransferase was solubilised from both preparations with optimal solubilisation being achieved at 0.5% (w/v) CHAPS concentrations. Solubilised GPATs were purified further using DE52 ion-exchange chromatography and Sephadex G-100 molecular exclusion chromatography. Purifications of up to about 70-fold were achieved. The purified GPATs showed a strong preference for palmitoyl-CoA compared to other acyl-CoA donors, in keeping with the importance of palmitate in palm oil. PMID:10664139

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

    PubMed

    Sternberg, Robin M; LeBlanc, Gerald A

    2006-06-30

    Exposure to tributyltin (TBT) has been causally associated with the global occurrence of a pseudohermaphroditic condition called imposex in neogastropod species. TBT elevates free testosterone levels in these organisms, and this upsurge in testosterone may be involved in the development of imposex. We investigated the ability of TBT to inhibit acyl coenzyme A:testosterone acyltransferase (ATAT) activity as well as microsomal acyl-coenzyme A:17beta-estradiol acyltransferase (AEAT) in a neogastropod, the eastern mud snail Ilyanassa obsoleta as a mechanism by which TBT elevates free testosterone. TBT significantly inhibited both ATAT and AEAT activities in vitro at toxicologically relevant in vivo concentrations. Kinetic analyses revealed that TBT is a competitive inhibitor of ATAT (K(i)= approximately 9microM) and is a weaker, noncompetitive inhibitor of AEAT (K(i)= approximately 31microM). ATAT and AEAT activities associated with different microsome preparations were significantly correlated, and 17beta-estradiol competitively inhibited the fatty acid esterification of testosterone suggesting that one enzyme is responsible for biotransforming both testosterone and 17beta-estradiol to their corresponding fatty acid esters. Overall, the results of this study supply the much-needed mechanistic support for the hypothesis that TBT elevates free testosterone in neogastropods by inhibiting their major regulatory process for maintaining free testosterone homeostasis-the fatty acid esterification of testosterone. PMID:16638618

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

    PubMed Central

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

    2014-01-01

    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. PMID:24821955

  17. The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

    PubMed Central

    De Smet, Cedric H.; Vittone, Elisa; Scherer, Max; Houweling, Martin; Liebisch, Gerhard; Brouwers, Jos F.; de Kroon, Anton I.P.M.

    2012-01-01

    The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta­king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings. PMID:22323296

  18. Castor Phospholipid:Diacylglycerol Acyltransferase Facilitates Efficient Metabolism of Hydroxy Fatty Acids in Transgenic Arabidopsis1[W][OA

    PubMed Central

    van Erp, Harrie; Bates, Philip D.; Burgal, Julie; Shockey, Jay; Browse, John

    2011-01-01

    Producing unusual fatty acids (FAs) in crop plants has been a long-standing goal of green chemistry. However, expression of the enzymes that catalyze the primary synthesis of these unusual FAs in transgenic plants typically results in low levels of the desired FA. For example, seed-specific expression of castor (Ricinus communis) fatty acid hydroxylase (RcFAH) in Arabidopsis (Arabidopsis thaliana) resulted in only 17% hydroxy fatty acids (HFAs) in the seed oil. In order to increase HFA levels, we investigated castor phospholipid:diacylglycerol acyltransferase (PDAT). We cloned cDNAs encoding three putative PDAT enzymes from a castor seed cDNA library and coexpressed them with RcFAH12. One isoform, RcPDAT1A, increased HFA levels to 27%. Analysis of HFA-triacylglycerol molecular species and regiochemistry, along with analysis of the HFA content of phosphatidylcholine, indicates that RcPDAT1A functions as a PDAT in vivo. Expression of RcFAH12 alone leads to a significant decrease in FA content of seeds. Coexpression of RcPDAT1A and RcDGAT2 (for diacylglycerol acyltransferase 2) with RcFAH12 restored FA levels to nearly wild-type levels, and this was accompanied by a major increase in the mass of HFAs accumulating in the seeds. We show the usefulness of RcPDAT1A for engineering plants with high levels of HFAs and alleviating bottlenecks due to the production of unusual FAs in transgenic oilseeds. PMID:21173026

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

    PubMed

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

    2016-06-01

    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]. (1)H 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

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

    PubMed

    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. PMID:24821955

  1. Functionally Divergent Alleles and Duplicated Loci Encoding an Acyltransferase Contribute to Acylsugar Metabolite Diversity in Solanum Trichomes[OPEN

    PubMed Central

    Schilmiller, Anthony L.; Moghe, Gaurav D.; Fan, Pengxiang; Ghosh, Banibrata; Ning, Jing; Jones, A. Daniel; Last, Robert L.

    2015-01-01

    Glandular trichomes from tomato (Solanum lycopersicum) and other species in the Solanaceae produce and secrete a mixture of O-acylsugars (aliphatic esters of sucrose and glucose) that contribute to insect defense. Despite their phylogenetic distribution and diversity, relatively little is known about how these specialized metabolites are synthesized. Mass spectrometric profiling of acylsugars in the S. lycopersicum x Solanum pennellii introgression lines identified a chromosome 11 locus containing a cluster of BAHD acyltransferases with one gene (named Sl-ASAT3) expressed in tip cells of type I trichomes where acylsugars are made. Sl-ASAT3 was shown to encode an acyl-CoA-dependent acyltransferase that catalyzes the transfer of short (four to five carbons) branched acyl chains to the furanose ring of di-acylsucrose acceptors to produce tri-acylsucroses, which can be further acetylated by Sl-ASAT4 (previously Sl-AT2). Among the wild tomatoes, diversity in furanose ring acyl chains on acylsucroses was most striking in Solanum habrochaites. S. habrochaites accessions from Ecuador and northern Peru produced acylsucroses with short (≤C5) or no acyl chains on the furanose ring. Accessions from central and southern Peru had the ability to add short or long (up to C12) acyl chains to the furanose ring. Multiple ASAT3-like sequences were found in most accessions, and their in vitro activities correlated with observed geographical diversity in acylsugar profiles. PMID:25862303

  2. Leishmania Dihydroxyacetonephosphate Acyltransferase LmDAT is Important for Ether Lipid Biosynthesis but not for the Integrity of Detergent Resistant Membranes

    PubMed Central

    Zufferey, Rachel; Al-Ani, Gada K.; Dunlap, Kara

    2009-01-01

    Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether-lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was colethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics. PMID:19720088

  3. Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities.

    PubMed

    Edlin, D A; Kille, P; Wilkinson, M D; Jones, H D; Harwood, J L

    2000-12-01

    We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme. PMID:11171169

  4. Identification of D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in Corynebacterium glutamicum.

    PubMed

    Lee, Jung-Hoon; Kim, Yong-Jae; Shin, Hee-Sung; Lee, Heung-Shick; Jin, Shouguang; Ha, Un-Hwan

    2016-06-01

    Expression of a putative acyltransferase encoded by NCgl- 0350 of Corynebacterium glutamicum is induced by cell-free culture fluids obtained from stationary-phase growth of both C. glutamicum and Pseudomonas aeruginosa, providing evidence for interspecies communication. Here, we further confirmed that such communication occurs by showing that acyltransferase expression is induced by culture fluid obtained from diverse Gram-negative and -positive bacterial strains, including Escherichia coli, Salmonella Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium sp. strain JC1, and Mycobacterium smegmatis. A homologous acyltransferase encoded by PA5238 of P. aeruginosa was also induced by fluids obtained from P. aeruginosa as well as other bacterial strains, as observed for NCgl0350 of C. glutamicum. Because C. glutamicum is difficult to study using molecular approaches, the homologous gene PA5238 of P. aeruginosa was used to identify PA5309 as an upstream regulator of expression. A homologous D-amino acid dehydrogenase encoded by NCgl- 2909 of C. glutamicum was cloned based on amino acid similarity to PA5309, and its role in the regulation of NCgl0350 expression was confirmed. Moreover, NCgl2909 played positive roles in growth of C. glutamicum. Thus, we identified a D-amino acid dehydrogenase as an upstream regulator of the autoinduction of a putative acyltransferase in C. glutamicum. PMID:27225460

  5. [Alcoholism and aging. 2. Alcoholic dementia or alcoholic cognitive impairment?].

    PubMed

    Pierucci-Lagha, Amira; Derouesné, Christian

    2003-12-01

    Chronic alcohol consumption results in considerable damage to many of the body's organs, and particularly to the brain. Beyond the confusional state occurring with acute intoxication or withdrawal, alcohol abuse is responsible of a constellation of neuropsychiatric syndromes including cognitive dysfunction, Wernicke-Korsakoff Syndrome, alcoholic cerebellar degeneration, Marchiafava-Bignami disease and alcohol-related dementia, ARD. ARD would account for nearly 20% of all admissions to state mental hospitals in the United-States. According to the DSM-IV, ARD is defined by a dementia associated with alcohol abuse. However, the concept of a dementia directly related to the neurotoxicity of alcohol for brain neurons is still a matter of debate. Several hypotheses have been proposed to explain the mechanisms of cognitive deficits related to chronic alcohol intoxication. This paper presents the epidemiological, neuropathological, neurochemical and clinical data on ARD. Alcoholism is responsible for cognitive deficits of various severity, which could be reversible or not with alcohol abstinence, but can also participate to the cognitive impairment related to other pathologies, such as Alzheimer disease. On account of this review, it is suggested that the term alcohol-related cognitive impairment should be more convenient than that of ARD, more restrictive and more confusing. Presently, there are no established treatment for alcohol-related cognitive impairment. Alcohol abstinence is a most important step. Psychosocial interventions are essential to support the patients in the daily life. PMID:15683959

  6. Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

    PubMed

    Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

    2013-08-01

    Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is

  7. Insomnia, alcoholism and relapse.

    PubMed

    Brower, Kirk J

    2003-12-01

    Insomnia and alcoholism are significantly associated in community surveys and patient samples. Insomnia occurs in 36-72% of alcoholic patients and may last for weeks to months after initiating abstinence from alcohol. Some correlates of insomnia in alcoholic patients are identical to those observed in non-alcoholic insomniacs, including anxiety and depression, tobacco smoking, and the use of alcohol to aid sleep. Other studies suggest that as the severity of alcoholism increases, so does the likelihood of insomnia in alcoholic patients. In the sleep laboratory, alcoholic patients who complain of insomnia have disrupted sleep continuity when compared to alcoholic patients without insomnia complaints. Recently sober alcoholics are also more likely than non-alcoholics to have sleep-disordered breathing and increased periodic leg movements, which might contribute to insomnia in some alcoholic patients. The co-occurrence of insomnia and alcoholism is clinically significant because alcoholism can exacerbate the adverse consequences of insomnia (e.g. mood changes and performance decrements) and because insomnia among patients entering treatment for alcoholism has been significantly associated with subsequent alcoholic relapse. Baseline polysomnographic correlates of subsequent relapse include prolonged sleep latency, decreased sleep efficiency and total sleep time, increased rapid eye movement sleep pressure, and decreased slow wave sleep. Whether treatment of insomnia in alcoholic patients reduces relapse rates is unknown, but preliminary treatment guidelines that accommodate the special characteristics of alcoholic patients are provided, with a goal to reduce daytime impairment and psychological distress. PMID:15018094

  8. Fetal Alcohol Syndrome

    MedlinePlus

    ... Conditions Frequently Asked Questions Español Condiciones Chinese Conditions Fetal Alcohol Syndrome Read in Chinese What is Fetal Alcohol Syndrome (FAS)? Fetal Alcohol Syndrome (FAS) describes changes in ...

  9. Fetal alcohol syndrome

    MedlinePlus

    Alcohol in pregnancy; Alcohol-related birth defects; Fetal alcohol effects; FAS ... the baby is in the womb and after birth Decreased muscle tone and ... Heart defects such as ventricular septal defect (VSD) or atrial ...

  10. Breath alcohol test

    MedlinePlus

    Alcohol test - breath ... There are various brands of breath alcohol tests. Each one uses a different method to test the level of alcohol in the breath. The machine may be electronic or manual. One ...