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Sample records for acyl-coa diacylglycerol acyltransferase

  1. 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...

  2. 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...

  3. 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...

  4. 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 ...

  5. 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.

  6. 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...

  7. 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.

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

    PubMed

    Chen, Jit Ern; Smith, Alison G

    2012-11-30

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

  9. 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...

  10. Identification of diacylglycerol acyltransferase inhibitors from Rosa centifolia petals.

    PubMed

    Kondo, Hidehiko; Hashizume, Kohjiro; Shibuya, Yusuke; Hase, Tadashi; Murase, Takatoshi

    2011-08-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final step of triacylglycerol (TAG) synthesis, and is considered as a potential target to control hypertriglyceridemia or other metabolic disorders. In this study, we found that the extract of rose petals suppressed TAG synthesis in cultured cells, and that the extract showed DGAT inhibitory action in a dose-dependent manner. Fractionation of the rose extract revealed that the DGAT inhibitory substances in the extract were ellagitannins; among them rugosin B, and D, and eusupinin A inhibited DGAT activity by 96, 82, and 84% respectively, at 10 μM. These substances did not inhibit the activities of other hepatic microsomal enzymes, glucose-6-phosphatase and HMG-CoA reductase, or pancreatic lipase, suggesting that ellagitannins inhibit DGAT preferentially. In an oral fat load test using mice, postprandial plasma TAG increase was suppressed by rose extract; TAG levels 2 h after the fat load were significantly lower in mice administered a fat emulsion containing rose extract than in control mice (446.3 ± 33.1 vs 345.3 ± 25.0 mg/dL, control vs rose extract group; P < 0.05). These results suggest that rose ellagitannins or rose extract could be beneficial in controlling lipid metabolism and used to improve metabolic disorders.

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

    PubMed

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

    2016-09-28

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

  12. Purification and properties of recombinant Brassica napus diacylglycerol acyltransferase 1.

    PubMed

    Caldo, Kristian Mark P; Greer, Michael S; Chen, Guanqun; Lemieux, M Joanne; Weselake, Randall J

    2015-03-12

    Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl-CoA-dependent triacylglycerol biosynthesis. Although the first DGAT1 gene was identified many years ago and the encoded enzyme catalyzes a key step in lipid biosynthesis, no detailed structure-function information is available on the enzyme due to difficulties associated with its purification. This study describes the purification of recombinant Brassica napus DGAT1 (BnaC.DGAT1.a) in active form through solubilization in n-dodecyl-β-D-maltopyranoside, cobalt affinity chromatography, and size-exclusion chromatography. Different BnaC.DGAT1.a oligomers in detergent micelles were resolved during the size-exclusion process. BnaC.DGAT1.a was purified 126-fold over the solubilized fraction and exhibited a specific activity of 26 nmol TAG/min/mg protein. The purified enzyme exhibited substrate preference for α-linolenoyl-CoA>oleoyl-CoA=palmitoyl-CoA>linoleoyl-CoA>stearoyl-CoA.

  13. Oleanane-type triterpenoids of Aceriphyllum rossii and their diacylglycerol acyltransferase-inhibitory activity.

    PubMed

    Seo, Jee-Hee; Kim, Mun-Ock; Han, Ah-Reum; Kwon, Eun-Bin; Kang, Myung Ji; Cho, Sungchan; Moon, Dong-Oh; Noh, Jung-Ran; Lee, Chul-Ho; Kim, Young-Soo; Lee, Hyun-Sun

    2015-02-01

    Six known triterpenoid compounds, 3-oxoolean-12-en-27-oic acid (1), gypsogenic acid (2), 3α-hydroxyolean-12-en-27-oic acid (3), 3β-hydroxyolean-12-en-27-oic acid (4), aceriphyllic acid A (5), and oleanolic acid (6), were isolated from the roots of Aceriphyllum rossii. Their chemical structures were determined by comparison with available (1)H-NMR and (13)C-NMR data on known compounds. All the isolated compounds were evaluated for inhibitory activity against human diacylglycerol acyltransferases 1 and 2. Most of the isolates exhibited a better inhibitory activity against diacylglycerol acyltransferase 2 (IC50: 11.6-44.2 µM) than against diacylglycerol acyltransferase 1 (IC50: 22.7-119.5 µM). In particular, compounds 1 and 5 showed strong inhibition efficacy towards diacylglycerol acyltransferases 1 and 2, and appeared to act competitively against oleoyl-CoA in vitro. The results also indicated that both compounds reduced newly synthesized triacylglycerol in HuTu80 and HepG2 cells. Oral administration of compound 1 significantly reduced postprandial triacylglycerol in mice following an oral lipid challenge. In conclusion, the current study indicates that compound 1 suppresses both de novo triacylglycerol biosynthesis and resynthesis through the inhibition of diacylglycerol acyltransferase activity, and therefore may be a useful agent for treating diseases associated with a high triacylglycerol level.

  14. 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...

  15. 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...

  16. 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...

  17. 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...

  18. Monoacylglycerol and diacylglycerol acyltransferases and the synthesis of neutral glycerides in Manduca sexta.

    PubMed

    Soulages, Jose L; Wu, Zengying; Firdaus, Sarah J; Mahalingam, Ramamurthy; Arrese, Estela L

    2015-07-01

    The insect fat body and the adipose tissue of vertebrates store fatty acids (FA) as triacylglycerols (TG). However, the fat body of most insects has the unique ability to rapidly produce and secrete large amounts of diacylglycerol (DG). Monoacylglycerol acyltransferase (MGAT), which catalyzes the synthesis of DG from MG, and a diacylglycerol acyltransferase (DGAT), which catalyzes the synthesis of TG from DG, are key enzymes in the metabolism of neutral glycerides. However, very little is known about these acyltransferases in insects. In the present study we have cloned two predicted MGATs and a DGAT from Manduca sexta and compared their sequences with predicted MGAT and DGAT homologs from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1. The apparent absence of a DGAT2 gene in insects would represent a major difference with vertebrates, which contain DGAT1 and DGAT2 genes. Insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. A number of conserved phosphorylation sites of potential physiological significance were identified among insect proteins and among insect and vertebrate proteins. DGAT1 and MGAT are expressed in fat body, midgut and ovaries. The relative rates of utilization of FAs for the synthesis of DG and TG correlated with the relative expression levels of MGAT and DGAT suggesting that regulation of the expression levels of these acyltransferases could be determining whether the fat body secretes DG or stores fatty acids as TG. The expression patterns of the acyltransferases suggest a role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.

  19. 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.

  20. 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

  1. Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

    PubMed

    Futatsugi, Kentaro; Kung, Daniel W; Orr, Suvi T M; Cabral, Shawn; Hepworth, David; Aspnes, Gary; Bader, Scott; Bian, Jianwei; Boehm, Markus; Carpino, Philip A; Coffey, Steven B; Dowling, Matthew S; Herr, Michael; Jiao, Wenhua; Lavergne, Sophie Y; Li, Qifang; Clark, Ronald W; Erion, Derek M; Kou, Kou; Lee, Kyuha; Pabst, Brandon A; Perez, Sylvie M; Purkal, Julie; Jorgensen, Csilla C; Goosen, Theunis C; Gosset, James R; Niosi, Mark; Pettersen, John C; Pfefferkorn, Jeffrey A; Ahn, Kay; Goodwin, Bryan

    2015-09-24

    The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

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

    PubMed

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

    2009-06-28

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

  3. 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

  4. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-17

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

  7. Evaluation of thiazole containing biaryl analogs as diacylglycerol acyltransferase 1 (DGAT1) inhibitors.

    PubMed

    Kadam, Kishorkumar S; Jadhav, Ravindra D; Kandre, Shivaji; Guha, Tandra; Reddy, M Mahesh Kumar; Brahma, Manoja K; Deshmukh, Nitin J; Dixit, Amol; Doshi, Lalit; Srinivasan, Shaila; Devle, Jayendra; Damre, Anagha; Nemmani, Kumar V S; Gupte, Amol; Sharma, Rajiv

    2013-07-01

    Biphenyl carboxylic acids, exemplified by compound 5, are known potent inhibitors of diacylglycerol acyltransferase, DGAT1, an enzyme involved in the final committed step of triglyceride biosynthesis. We have synthesized and evaluated 2-phenylthiazole, 4-phenylthiazole, and 5-phenylthiazole analogs as DGAT1 inhibitors. The 5-phenylthiazole series exhibited potent DGAT1 inhibition when evaluated using an in vitro enzymatic assay and an in vivo fat tolerance test in mice. Compound 33 (IC50 = 23 nM) exhibiting promising oral pharmacokinetic parameters (AUCinf = 7058 ng h/ml, T1/2 = 0.83 h) coupled with 87 percent reduction of plasma triglycerides in vivo may serve as a lead for developing newer anti-obesity agents.

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

    PubMed

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

    2006-12-27

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

  9. Therapeutic strategies for metabolic diseases: Small-molecule diacylglycerol acyltransferase (DGAT) inhibitors.

    PubMed

    Naik, Ravi; Obiang-Obounou, Brice W; Kim, Minkyoung; Choi, Yongseok; Lee, Hyun Sun; Lee, Kyeong

    2014-11-01

    Metabolic diseases such as atherogenic dyslipidemia, hepatic steatosis, obesity, and type II diabetes are emerging as major global health problems. Acyl-CoA:diacylglycerol acyltransferase (DGAT) is responsible for catalyzing the final reaction in the glycerol phosphate pathway of triglycerol synthesis. It has two isoforms, DGAT-1 and DGAT-2, which are widely expressed and present in white adipose tissue. DGAT-1 is most highly expressed in the small intestine, whereas DGAT-2 is primarily expressed in the liver. Therefore, the selective inhibition of DGAT-1 has become an attractive target with growing potential for the treatment of obesity and type II diabetes. Furthermore, DGAT-2 has been suggested as a new target for the treatment of DGAT-2-related liver diseases including hepatic steatosis, hepatic injury, and fibrosis. In view the discovery of drugs that target DGAT, herein we attempt to provide insight into the scope and further reasons for optimization of DGAT inhibitors.

  10. 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

  11. Diacylglycerol O-acyltransferase type-1 synthesizes retinyl esters in the retina and retinal pigment epithelium.

    PubMed

    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.

  12. 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.

  13. A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase.

    PubMed

    Seethala, Ramakrishna; Peterson, Tara; Dong, Jessica; Chu, Ching-Hsuen; Chen, Luping; Golla, Rajasree; Ma, Zhengping; Panemangalore, Reshma; Lawrence, R Michael; Cheng, Dong

    2008-12-15

    Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when (3)H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the (3)H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis-Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z' of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors.

  14. Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase.

    PubMed

    Saha, Saikat; Enugutti, Balaji; Rajakumari, Sona; Rajasekharan, Ram

    2006-08-01

    Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 +/- 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl- and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.

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

    PubMed

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

    2014-01-01

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

  17. 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.

  18. Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets.

    PubMed

    McFie, Pamela J; Jin, Youzhi; Banman, Shanna L; Beauchamp, Erwan; Berthiaume, Luc G; Stone, Scot J

    2014-09-01

    Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.

  19. Evaluation of food effect on the oral bioavailability of pradigastat, a diacylglycerol acyltransferase 1 inhibitor.

    PubMed

    Ayalasomayajula, Surya P; Meyers, Charles D; Yu, Jing; Kagan, Mark; Matott, Ralph; Pal, Parasar; Majumdar, Tapan; Su, Zhenzhong; Crissey, Anne; Rebello, Sam; Sunkara, Gangadhar; Chen, Jin

    2015-10-01

    Pradigastat, a diacylglycerol acyltransferase 1 inhibitor, is being developed for the treatment of familial chylomicronemia syndrome. The results of two studies that evaluated the effect of food on the oral bioavailability of pradigastat using randomized, open-label, parallel group designs in healthy subjects (n=24/treatment/study) are presented. In study 1, a single dose of 20 mg pradigastat was administered under the fasted condition or with a high-fat meal. In study 2, a single dose of 40 mg pradigastat was administered under the fasted condition or with a low- or high-fat meal. At the 20 mg dose, the pradigastat Cmax and AUClast increased by 38% and 41%, respectively, with a high-fat meal. When 40 mg pradigastat was administered with a low-fat meal, the Cmax and AUClast increased by 8% and 18%, respectively, whereas with a high-fat meal the increase was 20% and 18%, respectively. The population pharmacokinetic analysis with the pooled data from 13 studies indicated that administration of pradigastat with a meal resulted in an increase of 30% in both the Cmax and AUC parameters. Based on these results, food overall increased pradigastat exposure in the range of less than 40%, which is not considered clinically significant. Both 20 and 40 mg doses of pradigastat were well tolerated under fasted or fed conditions.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Surface plasmon resonance analysis of interactions between diacylglycerol acyltransferase and its interacting molecules.

    PubMed

    Kamisaka, Yasushi; Goto, Rie; Shibakami, Motonari; Yoshioka, Kyoko; Sato, Yukari

    2011-01-01

    To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.

  5. Two Clades of Type-1 Brassica napus Diacylglycerol Acyltransferase Exhibit Differences in Acyl-CoA Preference.

    PubMed

    Greer, Michael S; Pan, Xue; Weselake, Randall J

    2016-06-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to produce triacylglycerol, which is the main component of the seed oil of Brassica oilseed species. Phylogenetic analysis of the amino acid sequences encoded by four transcriptionally active DGAT1 genes from Brassica napus suggests that the gene forms diverged over time into two clades (I and II), with representative members in each genome (A and C). The majority of the amino acid sequence differences in these forms of DGAT1, however, reside outside of motifs suggested to be involved in catalysis. Despite this, the clade II enzymes displayed a significantly enhanced preference for linoleoyl-CoA when assessed using in-vitro enzyme assays with yeast microsomes containing recombinant enzyme forms. These findings contribute to our understanding of triacylglycerol biosynthesis in B. napus, and may advance our ability to engineer DGAT1s with desired substrate selectivity properties. PMID:27138895

  6. 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

  7. Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress.

    PubMed

    Liu, Xiao-Yu; Ouyang, Long-Ling; Zhou, Zhi-Gang

    2016-01-01

    In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress. PMID:27216435

  8. Phospholipid: diacylglycerol acyltransferase contributes to the conversion of membrane lipids into triacylglycerol in Myrmecia incisa during the nitrogen starvation stress

    PubMed Central

    Liu, Xiao-Yu; Ouyang, Long-Ling; Zhou, Zhi-Gang

    2016-01-01

    In addition to the Kennedy pathway for de novo biosynthesis, triacylglycerol (TAG), the most important stock for microalgae-based biodiesel production, can be synthesized by phospholipid: diacylglycerol acyltransferase (PDAT) that transfers an acyl group from phospholipids (PLs) to diacylglycerol (DAG). This study presents a novel gene that encodes PDAT from the green microalga Myrmecia incisa Reisigl H4301 (designated MiPDAT ). MiPDAT is localized on the plasma membrane (PM) via the agroinfiltration of tobacco leaves with a green fluorescent protein-fused construct. MiPDAT synthesizes TAG based on functional complementary experiments in the mutant yeast strain H1246 and the membrane lipid phosphatidylcholine (PC) is preferentially used as substrates as revealed by in vitro enzyme activity assay. The gradually increased transcription levels of MiPDAT in M. incisa during the cultivation under nitrogen starvation conditions is proposed to be responsible for the decrease and increase of the PC and TAG levels, respectively, as detected by liquid chromatography-mass spectrometry after 4 d of nitrogen starvation. In addition, the mechanism by which MiPDAT in this microalga uses PC to yield TAG is discussed. Accordingly, it is concluded that this PM-located PDAT contributes to the conversion of membrane lipids into TAG in M. incisa during the nitrogen starvation stress. PMID:27216435

  9. Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

    PubMed

    Liu, Jin; Han, Danxiang; Yoon, Kangsup; Hu, Qiang; Li, Yantao

    2016-04-01

    Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts. PMID:26919811

  10. Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

    PubMed

    Liu, Jin; Han, Danxiang; Yoon, Kangsup; Hu, Qiang; Li, Yantao

    2016-04-01

    Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts.

  11. Phospholipid:diacylglycerol acyltransferase-mediated triacylglycerol biosynthesis is crucial for protection against fatty acid-induced cell death in growing tissues of Arabidopsis.

    PubMed

    Fan, Jilian; Yan, Chengshi; Xu, Changcheng

    2013-12-01

    Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1-1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1-1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹⁴C-acetate labeling experiments showed that, compared with wild-type, tgd1-1 exhibits a 3.8-fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over-expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1-1. We also show that detached leaves of both pdat1-2 and tgd1-1 pdat1-2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA-induced cell death in fast-growing tissues of plants.

  12. Cardiomyocyte-specific loss of diacylglycerol acyltransferase 1 (DGAT1) reproduces the abnormalities in lipids found in severe heart failure.

    PubMed

    Liu, Li; Trent, Chad M; Fang, Xiang; Son, Ni-Huiping; Jiang, HongFeng; Blaner, William S; Hu, Yunying; Yin, Yu-Xin; Farese, Robert V; Homma, Shunichi; Turnbull, Andrew V; Eriksson, Jan W; Hu, Shi-Lian; Ginsberg, Henry N; Huang, Li-Shin; Goldberg, Ira J

    2014-10-24

    Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1(-/-) mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1(-/-)) mice. hDgat1(-/-) mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1(-/-) hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1(-/-) mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1(-/-)). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1(-/-) mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1(-/-) hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.

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

    PubMed Central

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

    2016-01-01

    The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications. PMID:27780240

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

    PubMed

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

    2015-12-01

    Synthesis of neutral lipids such as triacylglycerols (TAG) and wax esters (WE) is catalyzed in bacteria by wax ester synthase/diacylglycerol acyltransferase enzymes (WS/DGAT). We investigated the diversity of genes encoding this enzyme in contrasting natural environments from Patagonia (Argentina). The content of petroleum hydrocarbons in samples collected from oil-producing areas was measured. PCR-based analysis covered WS/DGAT occurrence in marine sediments and soil. No product was obtained in seawater samples. All clones retrieved from marine sediments affiliated with gammaproteobacterial sequences and within them, most phylotypes formed a unique cluster related to putative WS/DGAT belonging to marine OM60 clade. In contrast, soils samples contained phylotypes only related to actinomycetes. Among them, phylotypes affiliated with representatives largely or recently reported as oleaginous bacteria, as well as with others considered as possible lipid-accumulating bacteria based on the analysis of their annotated genomes. Our study shows for the first time that the environment could contain a higher variety of ws/dgat than that reported from bacterial isolates. The results of this study highlight the relevance of the environment in a natural process such as the synthesis and accumulation of neutral lipids. Particularly, both marine sediments and soil may serve as a useful source for novel WS/DGAT with biotechnological interest. PMID:26228353

  15. Diacylglycerol acyltransferase activity and triacylglycerol synthesis in germinating castor seed cotyledons.

    PubMed

    He, Xiaohua; Chen, Grace Q; Lin, Jiann-Tsyh; McKeon, Thomas A

    2006-03-01

    The central importance of storage lipid breakdown in providing carbon and energy during seed germination has been demonstrated by isolating the genes encoding the enzymes involved in FA beta-oxidation. In contrast, little is known about the ability of germinating seeds to synthesize TAG. We report that castor cotyledons are capable of TAG synthesis. The rate of incorporation of ricinoleic acid into TAG reached a peak at 7 d after imbibition (DAI) (1.14 nmol/h/mg) and decreased rapidly thereafter, but was sustained at 20 DAI in cotyledons and true leaves. The castor DAG acyltransferase (RcDGAT) mRNA and protein were expressed throughout seed germination at levels considerably enhanced from that in the dormant seed, thus indicating new expression. Significant degradation of the RcDGAT protein was observed after 7 DAI. The DGAT activity was found to be predominantly a function of the level of the intact RcDGAT protein, with the rate of TAG synthesis decreasing as degradation of the RcDGAT protein proceeded. A possible mechanism for the degradation of the RcDGAT protein is discussed. The induction of DGAT mRNA and protein, the capacity for TAG synthesis in vitro and in tissue slices, and the differing TAG composition of dormant seed TAG vs. cotyledonary TAG provide strong circumstantial evidence for active TAG synthesis by cotyledons. However, we have not yet determined the physiological significance of this capability.

  16. Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2

    PubMed Central

    Ahmad, Irshad; Sharma, Anil K.; Daniell, Henry; Kumar, Shashi

    2015-01-01

    Summary Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin-supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 106 cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro-fluorometric analysis of Nile red-stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α-linolenic acid, an essential omega-3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long-term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae. PMID:25403771

  17. Altered lipid composition and enhanced lipid production in green microalga by introduction of brassica diacylglycerol acyltransferase 2.

    PubMed

    Ahmad, Irshad; Sharma, Anil K; Daniell, Henry; Kumar, Shashi

    2015-05-01

    Higher lipid biosynthesis and accumulation are important to achieve economic viability of biofuel production via microalgae. To enhance lipid content, Chlamydomonas reinhardtii was genetically engineered with a key enzyme diacylglycerol acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral lipid biosynthesis. The transformed colonies harbouring aph7 gene, screened on hygromycin-supplemented medium, achieved transformation frequency of ~120 ± 10 colonies/1 × 10(6) cells. Transgene integration and expression were confirmed by PCR, Southern blots, staining lipid droplets, proteins and spectro-fluorometric analysis of Nile red-stained cells. The neutral lipid is a major class (over 80% of total lipids) and most significant requirement for biodiesel production; this was remarkably higher in the transformed alga than the untransformed control. The levels of saturated fatty acids in the transformed alga decreased to about 7% while unsaturated fatty acids increased proportionately when compared to wild type cells. Polyunsaturated fatty acids, especially α-linolenic acid, an essential omega-3 fatty acid, were enhanced up to 12% in the transformed line. Nile red staining confirmed formation of a large number of lipid globules in the transformed alga. Evaluation of long-term stability and vitality of the transgenic alga revealed that cryopreservation produced significantly higher quantity of lipid than those maintained continuously over 128 generations on solid medium. The overexpression of BnDGAT2 significantly altered the fatty acids profile in the transformed alga. Results of this study offer a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acids and neutral lipids for biofuel production in algae.

  18. Two types of soybean diacylglycerol acyltransferases are differentially involved in triacylglycerol biosynthesis and response to environmental stresses and hormones

    PubMed Central

    Chen, BeiBei; Wang, Junejie; Zhang, Gaoyang; Liu, Jiaqi; Manan, Sehrish; Hu, Honghong; Zhao, Jian

    2016-01-01

    Diacylglycerol acyltransferases (DGATs) play a key role in plant triacylglycerol (TAG) biosynthesis. Two type 1 and 2 DGATs from soybean were characterized for their functions in TAG biosynthesis and physiological roles. GmDGAT1A is highly expressed in seeds while GmDGAT2D is mainly expressed in flower tissues. They showed different expression patterns in response to biotic and abiotic stresses. GmDGAT2D was up-regulated by cold and heat stress and ABA signaling, and repressed by insect biting and jasmonate, whereas GmDGAT1A show fewer responses. Both GmDGAT1A and GmDGAT2D were localized to the endoplasmic reticulum and complemented the TAG deficiency of a yeast mutant H1246. GmDGAT2D-transgenic hairy roots synthesized more 18:2- or 18:1-TAG, whereas GmDGAT1A prefers to use 18:3-acyl CoA for TAG synthesis. Overexpression of both GmDGATs in Arabidopsis seeds enhanced the TAG production; GmDGAT2D promoted 18:2-TAG in wild-type but enhanced 18:1-TAG production in rod1 mutant seeds, with a decreased 18:3-TAG. However, GmDGAT1A enhanced 18:3-TAG and reduced 20:1-TAG contents. The different substrate preferences of two DGATs may confer diverse fatty acid profiles in soybean oils. While GmDGAT1A may play a role in usual seed TAG production and GmDGAT2D is also involved in usual TAG biosynthesis in other tissues in responses to environmental and hormonal cues. PMID:27345221

  19. Type II diacylglycerol acyltransferase from Claviceps purpurea with ricinoleic acid, a hydroxyl fatty acid of industrial importance, as preferred substrate.

    PubMed

    Mavraganis, Ioannis; Meesapyodsuk, Dauenpen; Vrinten, Patricia; Smith, Mark; Qiu, Xiao

    2010-02-01

    Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea. PMID

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

    PubMed Central

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

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics. PMID:26581109

  3. Discovery and optimization of adamantane carboxylic acid derivatives as potent diacylglycerol acyltransferase 1 inhibitors for the potential treatment of obesity and diabetes.

    PubMed

    Pagire, Suvarna H; Pagire, Haushabhau S; Lee, Gwi Bin; Han, Seo-Jung; Kwak, Hyun Jung; Kim, Ji Young; Kim, Ki Young; Rhee, Sang Dal; Ryu, Jeong Im; Song, Jin Sook; Bae, Myung Ae; Park, Mi-Jin; Kim, Dooseop; Lee, Duck Hyung; Ahn, Jin Hee

    2015-08-28

    We have developed a series of adamantane carboxylic acid derivatives exhibiting potent diacylglycerol acyltransferase 1 (DGAT1) inhibitory activities. Optimization of the series led to the discovery of E-adamantane carboxylic acid compound 43c, which showed excellent in vitro activity with an IC50 value of 5 nM against human and mouse DGAT1, also good druggability as well as microsomal stability and safety profiles such as hERG, CYP and cytotoxicity. Compound 43c significantly reduced plasma triglyceride levels in vivo (in rodents and zebrafish) and also showed bodyweight gain reduction and glucose area under curve (AUC) lowering efficacy in diet-induced obesity (DIO) mice.

  4. Identification of a pair of phospholipid:diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin.

    PubMed

    Pan, Xue; Siloto, Rodrigo M P; Wickramarathna, Aruna D; Mietkiewska, Elzbieta; Weselake, Randall J

    2013-08-16

    The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3(cis)(Δ9,12,15)) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.

  5. Type I Diacylglycerol Acyltransferase (MtDGAT1) from Macadamia tetraphylla: Cloning, Characterization, and Impact of Its Heterologous Expression on Triacylglycerol Composition in Yeast.

    PubMed

    Arroyo-Caro, José María; Mañas-Fernández, Aurora; Alonso, Diego López; García-Maroto, Federico

    2016-01-13

    Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired.

  6. Type I Diacylglycerol Acyltransferase (MtDGAT1) from Macadamia tetraphylla: Cloning, Characterization, and Impact of Its Heterologous Expression on Triacylglycerol Composition in Yeast.

    PubMed

    Arroyo-Caro, José María; Mañas-Fernández, Aurora; Alonso, Diego López; García-Maroto, Federico

    2016-01-13

    Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired. PMID:26666454

  7. Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii.

    PubMed

    Yoon, Kangsup; Han, Danxiang; Li, Yantao; Sommerfeld, Milton; Hu, Qiang

    2012-09-01

    Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.

  8. Modulation of phosphatidylcholine synthesis in vitro. Inhibition of diacylglycerol cholinephosphotransferase and lysophosphatidylcholine acyltransferase by centrophenoxine and neophenoxine.

    PubMed

    Parthasarathy, S; El-Rahman, A; Baumann, W J

    1981-08-24

    1,2-Diacyl-sn-glycerol : CDPcholine cholinephosphotransferase (EC 2.7.8.2) and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) activities of rat liver microsomes can be inhibited by centrophenoxine (N,N-dimethylaminoethyl p-chlorophenoxyacetate). This inhibition is brought about by the intact centrophenoxine molecule rather than by the products of hydrolysis. A nonhydrolyzable ether analog of centrophenoxine was synthesized (neophenoxine; N,N-dimethylaminoethyl p-chlorophenoxyethyl ether) and proved most effective in inhibiting the two routes of phosphatidylcholine biosynthesis. While 50% inhibition of the cholinephosphotransferase was attained at 5 mM neophenoxine, 50% inhibition of the acyltransferase required 0.6 mM neophenoxine levels only. Inhibition of the cholinephosphotransferase (Ki approximately 1.5 mM) and the acyltransferase (Ki approximately 1 mM) by neophenoxine was shown to be noncompetitive. Other membrane-bound enzymes, such as glucose-6-phosphatase, monoacylglycerol lipase, alkaline phosphatase or phospholipase A2 were not affected by the inhibitors. Because of this specificity, and because of the high affinity of the microsomal membrane for such agents, centrophenoxine and neophenoxine should prove useful for controlling phosphatidylcholine synthesis and for modulating the phosphatidylcholine deacylation-reacylation cycle.

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

    PubMed

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

    2014-10-15

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

  10. The effects of putative lipase and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase gene knockouts on triacylglycerol accumulation in Gordonia sp. KTR9.

    PubMed

    Indest, Karl J; Eberly, Jed O; Ringelberg, David B; Hancock, Dawn E

    2015-02-01

    Previously, we demonstrated triacylglycerol (TAG) accumulation and the in vivo ability to catalyze esters from exogenous short chain alcohol sources in Gordonia sp. strain KTR9. In this study, we investigated the effects that putative lipase (KTR9_0186) and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; KTR9_3844) gene knockouts had on TAG accumulation. Gene disruption of KTR9_0186 resulted in a twofold increase in TAG content in nitrogen starved cells. Lipase mutants subjected to carbon starvation, following nitrogen starvation, retained 75 % more TAGs and retained pigmentation. Transcriptome expression data confirmed the deletion of KTR9_0186 and identified the up-regulation of key genes involved in fatty acid degradation, a likely compensatory mechanism for reduced TAG mobilization. In vitro assays with purified KTR9_3844 demonstrated WS/DGAT activity with short chain alcohols and C16 and C18 fatty acid Co-As. Collectively, these results indicate that Gordonia sp. KTR9 has a suitable tractable genetic background for TAG production as well as the enzymatic capacity to catalyze fatty acid esters from short chain alcohols.

  11. Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation-inducible promoter.

    PubMed

    Iwai, Masako; Ikeda, Keiko; Shimojima, Mie; Ohta, Hiroyuki

    2014-08-01

    When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)- and phosphorus (P)-starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic-growth-phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation-dependent overexpressor of a Chlamydomonas type-2 diacylglycerol acyl-CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up-regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation-inducible promoter.

  12. The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

    PubMed Central

    Wang, Chao-Wen; Cheng, Yun-Hsin; Irokawa, Hayato; Hwang, Gi-Wook; Naganuma, Akira; Kuge, Shusuke

    2016-01-01

    Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins. PMID:27459103

  13. Altered Lipid Composition and Enhanced Nutritional Value of Arabidopsis Leaves following Introduction of an Algal Diacylglycerol Acyltransferase 2[C][W

    PubMed Central

    Sanjaya; Miller, Rachel; Durrett, Timothy P.; Kosma, Dylan K.; Lydic, Todd A.; Muthan, Bagyalakshmi; Koo, Abraham J.K.; Bukhman, Yury V.; Reid, Gavin E.; Howe, Gregg A.; Ohlrogge, John; Benning, Christoph

    2013-01-01

    Enhancement of acyl-CoA–dependent triacylglycerol (TAG) synthesis in vegetative tissues is widely discussed as a potential avenue to increase the energy density of crops. Here, we report the identification and characterization of Chlamydomonas reinhardtii diacylglycerol acyltransferase type two (DGTT) enzymes and use DGTT2 to alter acyl carbon partitioning in plant vegetative tissues. This enzyme can accept a broad range of acyl-CoA substrates, allowing us to interrogate different acyl pools in transgenic plants. Expression of DGTT2 in Arabidopsis thaliana increased leaf TAG content, with some molecular species containing very-long-chain fatty acids. The acyl compositions of sphingolipids and surface waxes were altered, and cutin was decreased. The increased carbon partitioning into TAGs in the leaves of DGTT2-expressing lines had little effect on transcripts of the sphingolipid/wax/cutin pathway, suggesting that the supply of acyl groups for the assembly of these lipids is not transcriptionally adjusted. Caterpillars of the generalist herbivore Spodoptera exigua reared on transgenic plants gained more weight. Thus, the nutritional value and/or energy density of the transgenic lines was increased by ectopic expression of DGTT2 and acyl groups were diverted from different pools into TAGs, demonstrating the interconnectivity of acyl metabolism in leaves. PMID:23417035

  14. JTT-553, a novel Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 inhibitor, improves glucose metabolism in diet-induced obesity and genetic T2DM mice.

    PubMed

    Tomimoto, Daisuke; Okuma, Chihiro; Ishii, Yukihito; Kobayashi, Akio; Ohta, Takeshi; Kakutani, Makoto; Imanaka, Tsuneo; Ogawa, Nobuya

    2015-09-01

    Type 2 diabetes mellitus (T2DM) arises primarily due to lifestyle factors and genetics. A number of lifestyle factors are known to be important in the development of T2DM, including obesity. JTT-553, a novel Acyl CoA:diacylglycerol acyltransferase 1 inhibitor, reduced body weight depending on dietary fat in diet-induced obesity (DIO) rats in our previous study. Here, the effect of JTT-553 on glucose metabolism was evaluated using body weight reduction in T2DM mice. JTT-553 was repeatedly administered to DIO and KK-A(y) mice. JTT-553 reduced body weight gain and fat weight in both mouse models. In DIO mice, JTT-553 decreased insulin, non-esterified fatty acid (NEFA), total cholesterol (TC), and liver triglyceride (TG) plasma concentrations in non-fasting conditions. JTT-553 also improved insulin-dependent glucose uptake in adipose tissues and glucose intolerance in DIO mice. In KK-A(y) mice, JTT-553 decreased glucose, NEFA, TC and liver TG plasma concentrations in non-fasting conditions. JTT-553 also decreased glucose, insulin, and TC plasma concentrations in fasting conditions. In addition, JTT-553 decreased TNF-α mRNA levels and increased GLUT4 mRNA levels in adipose tissues in KK-A(y) mice. These results suggest that JTT-553 improves insulin resistance in adipose tissues and systemic glucose metabolism through reductions in body weight.

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

    SciTech Connect

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

    2014-10-15

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

    Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae. Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 µm. Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wild-type DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans. PMID:27208257

  18. Acyl-CoA:Diacylglycerol Acyltransferase 1 Expression Level in the Hematopoietic Compartment Impacts Inflammation in the Vascular Plaques of Atherosclerotic Mice

    PubMed Central

    Vujic, Nemanja; Porter Abate, Jess; Schlager, Stefanie; David, Tovo; Koliwad, Suneil K.

    2016-01-01

    The final step of triacylglycerol synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferases (DGATs). We have previously shown that ApoE-/-Dgat1-/- mice are protected from developing atherosclerosis in association with reduced foam cell formation. However, the role of DGAT1, specifically in myeloid and other hematopoietic cell types, in determining this protective phenotype is unknown. To address this question, we reconstituted the bone marrow of irradiated Ldlr–/–mice with that from wild-type (WT→ Ldlr–/–) and Dgat1–/–(Dgat1–/–→ Ldlr–/–) donor mice. We noted that DGAT1 in the hematopoietic compartment exerts a sex-specific effect on systemic cholesterol homeostasis. However, both male and female Dgat1–/–→ Ldlr–/–mice had higher circulating neutrophil and lower lymphocyte counts than control mice, suggestive of a classical inflammatory phenotype. Moreover, specifically examining the aortae of these mice revealed that Dgat1–/–→ Ldlr–/–mice have atherosclerotic plaques with increased macrophage content. This increase was coupled to a reduced plaque collagen content, leading to a reduced collagen-to-macrophage ratio. Together, these findings point to a difference in the inflammatory contribution to plaque composition between Dgat1–/–→ Ldlr–/–and control mice. By contrast, DGAT1 deficiency did not affect the transcriptional responses of cultured macrophages to lipoprotein treatment in vitro, suggesting that the alterations seen in the plaques of Dgat1–/–→ Ldlr–/–mice in vivo do not reflect a cell intrinsic effect of DGAT1 in macrophages. We conclude that although DGAT1 in the hematopoietic compartment does not impact the overall lipid content of atherosclerotic plaques, it exerts reciprocal effects on inflammation and fibrosis, two processes that control plaque vulnerability. PMID:27223895

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

    PubMed

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

    2016-06-01

    Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 µm Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wild-type DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans. PMID:27208257

  20. Functional Characterization of Two Structurally Novel Diacylglycerol Acyltransferase2 Isozymes Responsible for the Enhanced Production of Stearate-Rich Storage Lipid in Candida tropicalis SY005

    PubMed Central

    Dey, Prabuddha; Chakraborty, Monami; Kamdar, Maulik R.; Maiti, Mrinal K.

    2014-01-01

    Diacylglycerol acyltransferase (DGAT) activity is an essential enzymatic step in the formation of neutral lipid i.e., triacylglycerol in all living cells capable of accumulating storage lipid. Previously, we characterized an oleaginous yeast Candida tropicalis SY005 that yields storage lipid up to 58% under a specific nitrogen-stress condition, when the DGAT-specific transcript is drastically up-regulated. Here we report the identification, differential expression and function of two DGAT2 gene homologues- CtDGAT2a and CtDGAT2b of this C. tropicalis. Two protein isoforms are unique with respect to the presence of five additional stretches of amino acids, besides possessing three highly conserved motifs known in other reported DGAT2 enzymes. Moreover, the CtDGAT2a and CtDGAT2b are characteristically different in amino acid sequences and predicted protein structures. The CtDGAT2b isozyme was found to be catalytically 12.5% more efficient than CtDGAT2a for triacylglycerol production in a heterologous yeast system i.e., Saccharomyces cerevisiae quadruple mutant strain H1246 that is inherently defective in neutral lipid biosynthesis. The CtDGAT2b activity rescued the growth of transformed S. cerevisiae mutant cells, which are usually non-viable in the medium containing free fatty acids by incorporating them into triacylglycerol, and displayed preferential specificity towards saturated acyl species as substrate. Furthermore, we document that the efficiency of triacylglycerol production by CtDGAT2b is differentially affected by deletion, insertion or replacement of amino acids in five regions exclusively present in two CtDGAT2 isozymes. Taken together, our study characterizes two structurally novel DGAT2 isozymes, which are accountable for the enhanced production of storage lipid enriched with saturated fatty acids inherently in C. tropicalis SY005 strain as well as in transformed S. cerevisiae neutral lipid-deficient mutant cells. These two genes certainly will be useful

  1. Type II Diacylglycerol Acyltransferase from Claviceps purpurea with Ricinoleic Acid, a Hydroxyl Fatty Acid of Industrial Importance, as Preferred Substrate ▿

    PubMed Central

    Mavraganis, Ioannis; Meesapyodsuk, Dauenpen; Vrinten, Patricia; Smith, Mark; Qiu, Xiao

    2010-01-01

    Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea. PMID

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2008-08-01

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

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

    PubMed

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

    2006-12-01

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

  5. Genome-Wide Analysis of PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) Genes in Plants Reveals the Eudicot-Wide PDAT Gene Expansion and Altered Selective Pressures Acting on the Core Eudicot PDAT Paralogs1[OPEN

    PubMed Central

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

    2015-01-01

    PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) is an enzyme that catalyzes the transfer of a fatty acyl moiety from the sn-2 position of a phospholipid to the sn-3-position of sn-1,2-diacylglyerol, thus forming triacylglycerol and a lysophospholipid. Although the importance of PDAT in triacylglycerol biosynthesis has been illustrated in some previous studies, the evolutionary relationship of plant PDATs has not been studied in detail. In this study, we investigated the evolutionary relationship of the PDAT gene family across the green plants using a comparative phylogenetic framework. We found that the PDAT candidate genes are present in all examined green plants, including algae, lowland plants (a moss and a lycophyte), monocots, and eudicots. Phylogenetic analysis revealed the evolutionary division of the PDAT gene family into seven major clades. The separation is supported by the conservation and variation in the gene structure, protein properties, motif patterns, and/or selection constraints. We further demonstrated that there is a eudicot-wide PDAT gene expansion, which appears to have been mainly caused by the eudicot-shared ancient gene duplication and subsequent species-specific segmental duplications. In addition, selection pressure analyses showed that different selection constraints have acted on three core eudicot clades, which might enable paleoduplicated PDAT paralogs to either become nonfunctionalized or develop divergent expression patterns during evolution. Overall, our study provides important insights into the evolution of the plant PDAT gene family and explores the evolutionary mechanism underlying the functional diversification among the core eudicot PDAT paralogs. PMID:25585619

  6. Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content.

    PubMed

    Winter, Andreas; Krämer, Wolfgang; Werner, Fabian A O; Kollers, Sonja; Kata, Srinivas; Durstewitz, Gregor; Buitkamp, Johannes; Womack, James E; Thaller, Georg; Fries, Ruedi

    2002-07-01

    DGAT1 encodes diacylglycerol O-acyltransferase (EC ), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein-Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof.

  7. Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content

    PubMed Central

    Winter, Andreas; Krämer, Wolfgang; Werner, Fabian A. O.; Kollers, Sonja; Kata, Srinivas; Durstewitz, Gregor; Buitkamp, Johannes; Womack, James E.; Thaller, Georg; Fries, Ruedi

    2002-01-01

    DGAT1 encodes diacylglycerol O-acyltransferase (EC 2.3.1.20), a microsomal enzyme that catalyzes the final step of triglyceride synthesis. It became a functional candidate gene for lactation traits after studies indicated that mice lacking both copies of DGAT1 are completely devoid of milk secretion, most likely because of deficient triglyceride synthesis in the mammary gland. Our mapping studies placed DGAT1 close to the region of a quantitative trait locus (QTL) on bovine chromosome 14 for variation in fat content of milk. Sequencing of DGAT1 from pooled DNA revealed significant frequency shifts at several variable positions between groups of animals with high and low breeding values for milk fat content in different breeds (Holstein–Friesian, Fleckvieh, and Braunvieh). Among the variants was a nonconservative substitution of lysine by alanine (K232A), with the lysine-encoding allele being associated with higher milk fat content. Haplotype analysis indicated the lysine variant to be ancestral. Two animals that were typed heterozygous (Qq) at the QTL based on marker-assisted QTL-genotyping were heterozygous for the K232A substitution, whereas 14 animals that are most likely qq at the QTL were homozygous for the alanine-encoding allele. An independent association study in Fleckvieh animals confirmed the positive effect of the lysine variant on milk fat content. We consider the nonconservative K232A substitution to be directly responsible for the QTL variation, although our genetic studies cannot provide formal proof. PMID:12077321

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

    PubMed Central

    Weselake, Randall J; Madhavji, Milan; Szarka, Steve J; Patterson, Nii A; Wiehler, William B; Nykiforuk, Cory L; Burton, Tracy L; Boora, Parveen S; Mosimann, Steven C; Foroud, Nora A; Thibault, Benjamin J; Moloney, Maurice M; Laroche, André; Furukawa-Stoffer, Tara L

    2006-01-01

    Background Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1–116)His6, with calculated molecular mass of 13,278 Da. Results BnDGAT1(1–116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1–116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1–116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1–116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. Conclusion Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER. PMID:17192193

  9. Effect of polymorphisms in the leptin, leptin receptor and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on bovine milk composition.

    PubMed

    Glantz, Maria; Lindmark Månsson, Helena; Stålhammar, Hans; Paulsson, Marie

    2012-02-01

    The relations between cow genetics and milk composition have gained a lot of attention during the past years, however, generally only a few compositional traits have been examined. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic polymorphism of β-casein (β-CN), κ-CN and β-lactoglobulin (β-LG) impact several bovine milk composition traits. Individual milk samples from the Swedish Red and Swedish Holstein breeds were analyzed for components in the protein, lipid, carbohydrate and mineral profiles. Cow alleles were determined on the following SNP: A1457G, A252T, A59V and C963T on the LEP gene, T945M on the LEPR gene and Nt984+8(A-G) on the DGAT1 gene. Additionally, genetic variants of β-CN, κ-CN and β-LG were determined. For both the breeds, the same tendency of minor allele frequency was found for all SNPs and protein genes, except on LEPA1457G and LEPC963T. This study indicated significant (P<0·05) associations between the studied SNPs and several compositional parameters. Protein content was influenced by LEPA1457G (G>A) and LEPC963T (T>C), whereas total Ca, ionic Ca concentration and milk pH were affected by LEPA1457G, LEPA59V, LEPC963T and LEPRT945M. However, yields of milk, protein, CN, lactose, total Ca and P were mainly affected by β-CN (A2>A1) and κ-CN (A>B>E). β-LG was mainly associated with whey protein yield and ionic Ca concentration (A>B). Thus, this study shows possibilities of using these polymorphisms as markers within genetic selection programs to improve and adjust several compositional parameters.

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

    PubMed Central

    Kuehn, Christa; Edel, Christian; Weikard, Rosemarie; Thaller, Georg

    2007-01-01

    Background Substantial gene substitution effects on milk production traits have formerly been reported for alleles at the K232A and the promoter VNTR loci in the bovine acylCoA-diacylglycerol-acyltransferase 1 (DGAT1) gene by using data sets including sires with accumulated phenotypic observations of daughters (breeding values, daughter yield deviations). However, these data sets prevented analyses with respect to dominance or parent-of-origin effects, although an increasing number of reports in the literature outlined the relevance of non-additive gene effects on quantitative traits. Results Based on a data set comprising German Holstein cows with direct trait measurements, we first confirmed the previously reported association of DGAT1 promoter VNTR alleles with milk production traits. We detected a dominant mode of effects for the DGAT1 K232A and promoter VNTR alleles. Namely, the contrasts between the effects of heterozygous individuals at the DGAT1 loci differed significantly from the midpoint between the effects for the two homozygous genotypes for several milk production traits, thus indicating the presence of dominance. Furthermore, we identified differences in the magnitude of effects between paternally and maternally inherited DGAT1 promoter VNTR – K232A haplotypes indicating parent-of-origin effects on milk production traits. Conclusion Non-additive effects like those identified at the bovine DGAT1 locus have to be accounted for in more specific QTL detection models as well as in marker assisted selection schemes. The DGAT1 alleles in cattle will be a useful model for further investigations on the biological background of non-additive effects in mammals due to the magnitude and consistency of their effects on milk production traits. PMID:17892573

  11. Effect of polymorphisms in the leptin, leptin receptor, and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes and genetic polymorphism of milk proteins on cheese characteristics.

    PubMed

    Glantz, M; Lindmark Månsson, H; Stålhammar, H; Paulsson, M

    2011-07-01

    Cheese production has increased worldwide during the last decade and is expected to increase within the coming decade as well. Despite this, the relations between cow genetics and cheese characteristics are not fully known. The aim of this study was to determine if polymorphisms in the leptin (LEP), leptin receptor (LEPR), and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) genes as well as genetic variants of β-casein (β-CN), κ-CN, and β-lactoglobulin (β-LG) affect technological properties important for cheese production and, hence, could act as genetic makers for cheese quality. Individual milk samples from the Swedish Red and the Swedish Holstein breeds were analyzed for sizes of CN micelles and fat globules as well as rennet-induced gel strength, gelation time, and yield stress. Model cheeses were produced to study yield, hardness, and pH of the cheeses. The A1457G, A252T, A59V, and C963T single nucleotide polymorphisms (SNP) were analyzed on the LEP gene, the T945M SNP on the LEPR gene, and the Nt984+8(A-G) SNP on the DGAT1 gene. In addition, genetic variants of β-CN, κ-CN, and β-LG were determined. The results indicate that technological properties were influenced by the LEPR(T945M) polymorphism, which had an association with gel strength, yield stress, and cheese hardness (T > C). However, also LEP(A252T) was shown to affect gel strength (T > A), whereas the LEP(A59V) had an effect on fat globule size (T > C). For the milk protein genes, favorable effects were found for the A and B variants of β-LG and κ-CN, respectively, on gel strength, gelation time, and yield stress. In addition, the B variant of κ-CN was shown to be associated with smaller CN micelles than the A variant. Thus, the results demonstrate potential genetic markers for cheese characteristics. However, milk composition traits also affected the obtained results, thus making it necessary to thoroughly assess the different aspects regarding the influence of gene effects on

  12. 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.

  13. 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

  14. 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

  15. Inhibition of diacylglycerol kinases as a physiological way to promote diacylglycerol signaling.

    PubMed

    Baldanzi, Gianluca

    2014-05-01

    Diacylglycerol is a key regulator of cell physiology, controlling the membrane recruitment and activation of signaling molecules. Accordingly, diacylglycerol generation and metabolism are strictly controlled, allowing for localized regulation of its concentration. While the increased production of diacylglycerol upon receptor triggering is well recognized, the modulation of diacylglycerol metabolism by diacylglycerol kinases (DGKs) is less characterized. Some agonists induce DGK activation and recruitment to the plasma membrane, promoting diacylglycerol metabolism to phosphatidic acid. Conversely, several reports indicate that signaling pathways that selectively inhibits DGK isoforms can enhance cellular diacylglycerol levels and signal transduction. For example, the impairment of DGKθ activity by RhoA binding to the catalytic domain represents a conserved mechanism controlling diacylglycerol signaling from Caenorhabditis elegans motoneurons to mammalian hepatocytes. Similarly, DGKα activity is inhibited in lymphocytes by TCR signaling, thus contributing to a rise in diacylglycerol concentration for downstream signaling. Finally, DGKμ activity is inhibited by ischemia-reperfusion-generated reactive oxygen species in airway endothelial cells, promoting diacylglycerol-mediated ion channel opening and edema. In those systems, DGKs provide a gatekeeper function by blunting diacylglycerol levels or possibly establishing permissive domains for diacylglycerol signaling. In this review, I discuss the possible general relevance of DGK inhibition to enhanced diacylglycerol signaling.

  16. Inhibited insulin signaling in mouse hepatocytes is associated with increased phosphatidic acid but not diacylglycerol.

    PubMed

    Zhang, Chongben; Hwarng, Gwen; Cooper, Daniel E; Grevengoed, Trisha J; Eaton, James M; Natarajan, Viswanathan; Harris, Thurl E; Coleman, Rosalind A

    2015-02-01

    Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes.

  17. Membrane Topology of Hedgehog Acyltransferase*

    PubMed Central

    Matevossian, Armine; Resh, Marilyn D.

    2015-01-01

    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

  18. Diacylglycerol kinase η1 is a high affinity isozyme for diacylglycerol.

    PubMed

    Komenoi, Suguru; Takemura, Fumika; Sakai, Hiromichi; Sakane, Fumio

    2015-05-01

    Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied.

  19. DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development[W][OA

    PubMed Central

    Zhang, Meng; Fan, Jilian; Taylor, David C.; Ohlrogge, John B.

    2009-01-01

    Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis. PMID:20040537

  20. Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors.

    PubMed

    Imbriglio, Jason E; Shen, Dong-Ming; Liang, Rui; Marby, Ken; You, Ming; Youm, Hye Won; Feng, Zhe; London, Clare; Xiong, Yusheng; Tata, Jim; Verras, Andreas; Garcia-Calvo, Margarita; Song, Xuelei; Addona, George H; McLaren, Dave G; He, Timothy; Murphy, Beth; Metzger, Dan E; Salituro, Gino; Deckman, Diana; Chen, Qing; Jin, Xiaoling; Stout, Steven J; Wang, Sheng-Ping; Wilsie, Larissa; Palyha, Oksana; Han, Seongah; Hubbard, Brian K; Previs, Stephen F; Pinto, Shirly; Taggart, Andrew

    2015-12-10

    DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

  1. Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 expressi...

  2. Discovery of diamide compounds as diacylglycerol acyltransferase 1 (DGAT1) inhibitors.

    PubMed

    Nakajima, Katsumasa; April, Myriam; Brewer, Jason T; Daniels, Thomas; Forster, Cornelia J; Gilmore, Thomas A; Jain, Monish; Kanter, Aaron; Kwak, Youngshin; Li, Jingzhou; McQuire, Les; Serrano-Wu, Michael H; Streeper, Ryan; Szklennik, Paul; Thompson, James; Wang, Bing

    2016-02-15

    Diamide compounds were identified as potent DGAT1 inhibitors in vitro, but their poor molecular properties resulted in low oral bioavailability, both systemically and to DGAT1 in the enterocytes of the small intestine, resulting in a lack of efficacy in vivo. Replacing an N-alkyl group on the diamide with an N-aryl group was found to be an effective strategy to confer oral bioavailability and oral efficacy in this lipophilic diamide class of inhibitors.

  3. Synthesis and multiparametric evaluation of thiadiazoles and oxadiazoles as diacylglycerol acyltransferase type 1 inhibitors.

    PubMed

    Mougenot, Patrick; Namane, Claudie; Fett, Eykmar; Goumy, Florence; Dadji-Faïhun, Rommel; Langot, Gwladys; Monseau, Catherine; Onofri, Bénédicte; Pacquet, François; Pascal, Cécile; Crespin, Olivier; Ben-Hassine, Majdi; Ragot, Jean-Luc; Van-Pham, Thao; Philippo, Christophe; Chatelain-Egger, Florence; Péron, Philippe; Le Bail, Jean-Christophe; Guillot, Etienne; Chamiot-Clerc, Philippe; Chabanaud, Marie-Aude; Pruniaux, Marie-Pierre; Ménegotto, Jérôme; Schmidt, Friedemann; Venier, Olivier; Viviani, Fabrice; Nicolai, Eric

    2016-01-01

    Chemical modulation of a formerly disclosed DGAT-1 inhibitor resulted in the identification of a compound with a suitable profile for preclinical development. Optimisation of solubility is discussed and a PK/PD study is presented.

  4. Leukocyte chemoattractant activity of diacylglycerol

    SciTech Connect

    Wright, T.M.; Hoffman, R.D.; Nishijima, J.; Shin, H.S.

    1986-03-05

    Phosphatidylinositol breakdown with the generation of 1,2-diacylglycerol (1,2-DG) and inositol phosphates occurs in response to receptor mediated stimulation of lymphocytes and polymorphonuclear neutrophils (PMN). In the authors attempt to demonstrate the direct role of 1,2-DG in cell migration, they have found 1,2 dioctanoyl glycerol (1,2-C8DG) to be a chemoattractant for 6C3HED, a mouse thymic lymphoma, and human peripheral blood PMN's. The chemoattractant activity for both cell types was observed at concentrations from 0.5 to 10mM in an under agarose assay. The maximum effect of 1,2-C8DG on 6C3HED cells was similar to that of 1mM lysophosphatidylcholine and the maximum effect of 1,2-C8DG on PMN's was similar to that of 10/sup -7/M f-met-leu-phe. Other 1,2-DG's with acyl chains ranging from 6 to 18 carbons in length and 1-oleoyl-2-acetyl-glycerol were also chemoattractants for 6C3HED, although their activities were less than 1,2-C8DG. In addition, phorbol myristate acetate (PMA), another activator of protein kinase C, was a chemoattractant for 6C3HED and human PMN's. PMA was more potent than 1,2-C8DG for both 6C3HED and PMN's with chemoattractant activity in the range of 30nM to 1..mu..M. These studies support the direct role of 1,2-DG in the transduction of chemotactic stimuli in leukocytes and further suggest that the formation of diacylglycerol represents a common step in the migratory responses of lymphoid and myeloid cells.

  5. Attenuation of diacylglycerol second messengers

    SciTech Connect

    Bishop, W.R.; Ganong, B.R.; Bell, R.M.

    1986-05-01

    Diacylglycerol(DAG) derived from phosphatidylinositol activates protein kinase C in agonist-stimulated cells. At least two pathways may contribute to the attenuation of the DAG signal: (1) phosphorylation to phosphatidic acid(PA) by DAG kinase(DGK), and (2) deacylation by DAG and monoacylglycerol lipases. A number of DAG analogs were tested as substrates and inhibitors of partially purified pig brain DGK. Two analogs were potent inhibitors in vitro, 1-monooleoylglycerol(MOG,K/sub I/ = 91 ..mu..M) and diotanoylethyleneglycol (diC/sub 8/EG, K/sub I/ = 58 ..mu..M). These compounds were tested in human platelets. DiC/sub 8/EG inhibited (70 - 100%) (/sup 32/P/sub i/) incorporation into PA in thrombin-stimulated platelets. Under these conditions the DAG signal was somewhat long-lived but was still metabolized, presumably by the lipase pathway. MOG treatment elevated DAG levels up to 4-fold in unstimulated platelets. The DAG formed was in a pool where it did not activate protein kinase C. Thrombin-stimulation of MOG-treated platelets resulted in DAG levels 10-fold higher than control platelets. This appears to be due to the inability of these platelets to metabolize agonist-linked DAG via the lipase pathway. The development of specific inhibitors of DAG kinase and DAG lipase, in conjunction with mass quantification of DAG levels as used here, will provide further insights into the regulation of DAG second messengers.

  6. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  7. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  8. 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

  9. The Immunomodulatory Functions of Diacylglycerol Kinase ζ

    PubMed Central

    Singh, Brenal K.; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock.

  10. The Immunomodulatory Functions of Diacylglycerol Kinase ζ

    PubMed Central

    Singh, Brenal K.; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock. PMID:27656643

  11. Assay and Inhibition of Diacylglycerol Lipase Activity

    PubMed Central

    Johnston, Meghan; Bhatt, Shachi R.; Sikka, Surina; Mercier, Richard W.; West, Jay M.; Makriyannis, Alexandros; Gatley, S. John; Duclos, Richard I.

    2012-01-01

    A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-14C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-14C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-14C]arachidonic acid. PMID:22738638

  12. The Immunomodulatory Functions of Diacylglycerol Kinase ζ.

    PubMed

    Singh, Brenal K; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock. PMID:27656643

  13. The Immunomodulatory Functions of Diacylglycerol Kinase ζ.

    PubMed

    Singh, Brenal K; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock.

  14. 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

  15. 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.

  16. Evidence for a compartmentation of brain microsomal diacylglycerol

    SciTech Connect

    Binaglia, L.; Roberti, R.; Vecchini, A.; Porcellati, G.

    1982-09-01

    Phosphatidylcholine synthesis from CDP-(methyl-/sup 14/C)choline and membrane-bound diacyl-(U-/sup 14/C)-sn-glycerol, formed through the glycerol phosphate pathway, has been examined in vitro in rat brain microsomes. When labeled diacylglycerol was incubated in the presence of unlabeled CDP-choline, the rate of phospholipid labeling looked very different from that measured in incubations of unlabeled diacylglycerol with CDP-(methyl-/sup 14/C)choline. Evidence is given that diacylglycerol formed through the glycerol phosphate pathway belongs to a metabolic pool separate from the bulk membrane diacylglycerol.

  17. 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

  18. Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system.

    PubMed

    Besterman, J M; Pollenz, R S; Booker, E L; Cuatrecasas, P

    1986-12-01

    Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500- to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.

  19. Diacylglycerol Kinase Inhibition and Vascular Function.

    PubMed

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction. PMID:21547002

  20. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ.

    PubMed

    Ard, Ryan; Mulatz, Kirk; Pomoransky, Julia L; Parks, Robin J; Trinkle-Mulcahy, Laura; Bell, John C; Gee, Stephen H

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.

  1. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ

    PubMed Central

    Pomoransky, Julia L.; Parks, Robin J.; Trinkle-Mulcahy, Laura; Bell, John C.; Gee, Stephen H.

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis. PMID:26701304

  2. 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.

  3. Solvent-free lipase-catalyzed preparation of diacylglycerols.

    PubMed

    Weber, Nikolaus; Mukherjee, Kumar D

    2004-08-25

    Various methods have been applied for the enzymatic preparation of diacylglycerols that are used as dietary oils for weight reduction in obesity and related disorders. Interesterification of rapeseed oil triacylglycerols with commercial preparations of monoacylglycerols, such as Monomuls 90-O18, Mulgaprime 90, and Nutrisoft 55, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) in vacuo at 60 degrees C led to extensive (from 60 to 75%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with Nutrisoft, catalyzed by Lipozyme RM in vacuo at 60 degrees C, also led to extensive (from 60 to 70%) formation of diacylglycerols. Esterification of rapeseed oil fatty acids with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme RM and lipases from Thermomyces lanuginosus (Lipozyme TL IM) and Candida antarctica (lipase B, Novozym 435), also provided diacylglycerols, however, to a lower extent (40-45%). Glycerolysis of rapeseed oil triacylglycerols with glycerol in vacuo at 60 degrees C, catalyzed by Lipozyme TL and Novozym 435, led to diacylglycerols to the extent of diacylglycerol oils) containing 66-70% diacylglycerols.

  4. 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

  5. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    SciTech Connect

    Cabot, M.C.

    1984-08-30

    Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56/sup 0/C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 ..mu..M range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the (/sup 14/C)dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of (/sup 14/)dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols. 44 references, 3 figures, 1 table.

  6. Reorganization of cellular retinol-binding protein type 1 and lecithin:retinol acyltransferase during retinyl ester biosynthesis

    PubMed Central

    Jiang, Weiya; Napoli, Joseph L.

    2012-01-01

    Background Cellular retinol-binding protein, type 1 (Crbp1), chaperones retinyl ester (RE) biosynthesis catalyzed by lecithin:retinol acyltransferase (LRAT). Methods We monitored the subcellular loci of LRAT and Crbp1 before and during RE biosynthesis, and compared the results to diacylglycerol:acyltransferase type 2 (DGAT2) during triacylglycerol biosynthesis in three cell lines: COS7, CHO and HepG2. Results Before initiation of RE biosynthesis, LRAT distributed throughout the endoplasmic reticulum (ER), similar to DGAT2, and Crpb1 localized with mitochondria associated membranes (MAM), surrounded by LRAT. Upon initiating RE biosynthesis in cells transfected with low amounts of vector to simulate physiological expression levels, Crpb1 remained with MAM, and both Crbp1 and MAM re-localized with LRAT. LRAT formed rings around the growing lipid droplets. LRAT activity was higher in these rings relative to the general ER. LRAT-containing rings colocalized with the lipid-droplet surface proteins, desnutrin/adipose triglyceride lipase and perilipin 2. Colocalization with lipid droplets required the 38 N-terminal amino acid residues of LRAT, and specifically K36 and R38. Formation of rings around the growing lipid droplets did not require functional microtubules. General significance These data indicate a relationship between LRAT and Crbp1 during RE biosynthesis in which MAM-associated Crpb1 and LRAT colocalize, and both surround the growing RE-containing lipid droplet. The N-terminus of LRAT, especially K36 and R38, are essential to colocalization with the lipid droplet. PMID:22498138

  7. Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization.

    PubMed

    Take, Kazumi; Mochida, Taisuke; Maki, Toshiyuki; Satomi, Yoshinori; Hirayama, Megumi; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Sato, Kenjiro; Kitazaki, Tomoyuki; Takekawa, Shiro

    2016-01-01

    Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders. PMID:26938273

  8. Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization

    PubMed Central

    Take, Kazumi; Mochida, Taisuke; Maki, Toshiyuki; Satomi, Yoshinori; Hirayama, Megumi; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Sato, Kenjiro; Kitazaki, Tomoyuki; Takekawa, Shiro

    2016-01-01

    Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders. PMID:26938273

  9. 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.

  10. Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis[C][W

    PubMed Central

    Fan, Jilian; Yan, Chengshi; Roston, Rebecca; Shanklin, John

    2014-01-01

    Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal β-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves. PMID:25293755

  11. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

    PubMed Central

    Owen, M R; Corstorphine, C C; Zammit, V A

    1997-01-01

    The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed. PMID:9173878

  12. Locust adipokinetic hormones mobilize diacylglycerols selectively.

    PubMed

    Tomcala, Ales; Bártů, Iva; Simek, Petr; Kodrík, Dalibor

    2010-05-01

    The diacylglycerols (DG) molecular species and their fatty acid (FA) composition were investigated by electrospray mass spectrometry (ESI-MS) and by gas chromatography with flame ionisation detection (GC-FID) in haemolymph of Locusta migratoria after application of adipokinetic hormones Locmi-AKH-I, -II and -III. The analyses showed (1) a heterogeneous distribution of individual DGs in haemolymph after the hormone application. The results revealed that mobilization of the DGs is molecular species-specific with the highest proportion of 34:1 DG (16:0/18:1 - mw 594Da) for all Locmi-AKHs bearing palmitic acid (C16:0) and oleic acid (C18:1) residues, and forming about 20% of the total mobilized DG content. (2) Analysis of fat body triacylglycerols revealed that all Locmi-AKHs mobilize the DGs selectively with the preference of those possessing the C18 and C16 FAs. The fat body FAs with carbon chain longer than 18 did not participate in the mobilization. (3) A distribution of FAs in the DG structures obtained by LC/ESI-MS, and FA analysis by GC-FID after transmethylation indicated a certain degree of Locmi-AKH selectivity toward the mobilized DGs and hence the FAs. The Locmi-AKH-I significantly prefers mobilization of DGs containing unsaturated FAs, while Locmi-AKH-II and -III prefer mobilization of saturated FAs. PMID:20139028

  13. Diacylglycerol kinase α establishes T cell polarity by shaping diacylglycerol accumulation at the immunological synapse.

    PubMed

    Chauveau, Anne; Le Floc'h, Audrey; Bantilan, Niels S; Koretzky, Gary A; Huse, Morgan

    2014-08-26

    Polarization of the T cell microtubule-organizing center (MTOC) to the immunological synapse between the T cell and an antigen-presenting cell (APC) maintains the specificity of T cell effector responses by enabling directional secretion toward the APC. The reorientation of the MTOC is guided by a sharp gradient of the second messenger diacylglycerol (DAG), which is centered at the immunological synapse. We used a single-cell photoactivation approach to demonstrate that diacylglycerol kinase α (DGK-α), which catalyzes the conversion of DAG to phosphatidic acid, determined T cell polarity by limiting the diffusion of DAG. DGK-α-deficient T cells exhibited enlarged accumulations of DAG at the immunological synapse, as well as impaired reorientation of the MTOC. In contrast, T cells lacking the related isoform DGK-ζ did not display polarization defects. We also found that DGK-α localized preferentially to the periphery of the immunological synapse, suggesting that it constrained the area over which DAG accumulated. Phosphoinositide 3-kinase activity was required for the peripheral localization pattern of DGK-α, which suggests a link between DAG and phosphatidylinositol signaling during T cell activation. These results reveal a previously unappreciated function of DGK-α and provide insight into the mechanisms that determine lymphocyte polarity.

  14. Pleiotropic effects of acyltransferases on various virulence-related phenotypes of Pseudomonas aeruginosa.

    PubMed

    Yeom, Doo Hwan; Kim, Soo-Kyoung; Lee, Mi-Nan; Lee, Joon-Hee

    2013-08-01

    Pseudomonas aeruginosa, an opportunistic pathogen causing various infections, expresses various virulence factors under the control of quorum sensing (QS), a cell density-sensing mechanism. Because the major signal molecules of QS are acyl homoserine lactones (acyl-HSLs), acyltransferases, the enzymes that act upon acyl group transfer could affect the QS signaling and QS-related virulence phenotypes. In this study, we overexpressed acyltransferases of P. aeruginosa and screened them for the activity influencing the QS and QS-related virulence phenotypes. Among seven acyltransferases tested in this study, two acyltransferases, PA3984 (apolipoprotein N-acyltransferase) and PA2537 (putative acyltransferase), significantly affected both growth of P. aeruginosa and the activity of LasR, a major QS regulator, when overexpressed. These acyltransferases also reduced virulence and swarming motility of P. aeruginosa. The other acyltransferase, PA3646 (UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase), reduced the LasR activity, swarming motility, protease production and virulence without any influence on growth. These effects by PA3646 over-expression were caused by less production of QS signal. PA3644 (UDP-N-acetylglucosamine acyltransferase) enhanced biofilm formation and swarming motility with no effect on the growth and QS activity. These results suggest that acyltransferases may be an important factor regulating the cellular activity about virulence-related phenotypes. PMID:23848169

  15. Diacylglycerol Kinase ϵ Is Selective for Both Acyl Chains of Phosphatidic Acid or Diacylglycerol*

    PubMed Central

    Lung, Michael; Shulga, Yulia V.; Ivanova, Pavlina T.; Myers, David S.; Milne, Stephen B.; Brown, H. Alex; Topham, Matthew K.; Epand, Richard M.

    2009-01-01

    The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase ϵ (DGKϵ) has an important role in this cycle. DGKϵ is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGKϵ by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGKϵ does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGKϵ regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGKϵ remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell. PMID:19744926

  16. Diacylglycerol Kinase from Suspension Cultured Plant Cells 1

    PubMed Central

    Wissing, Josef; Heim, Sabina; Wagner, Karl G.

    1989-01-01

    Diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from suspension-cultured Catharanthus roseus cells was extracted from a membrane fraction with 0.6% Triton X-100 and 150 millimolar NaCl and was purified about 900-fold by DEAE-cellulose, blue Sepharose, gel permeation, and phenyl-Sepharose chromatography. The enzyme is obviously membrane bound as activity in the cytosol could not be detected. In the presence of detergents such as Triton X-100 (3-[3-cholamidopropyl]dimethylamino)-1-propanesulfonate (Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by gel filtration. In glycerol density gradients, the enzyme sedimented slightly more slowly than bovine serum albumin, indicating a molecular weight of less than 68,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzyme activity could be assigned to a protein of 51,000 daltons. As found previously for bacterial and animal diacylglycerol kinases, the purified enzyme was completely devoid of activity without the addition of phospholipids or deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts of detergent were inhibitory. The enzyme needs divalent cations for activity, with Mg2+ ions being the most effective. Apparent Km values for ATP and diacylglycerol were determined as 100 and 250 micromolar, respectively. PMID:16666963

  17. 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.

  18. 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.

  19. 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

  20. Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes.

    PubMed

    Soupene, Eric; Fyrst, Henrik; Kuypers, Frans A

    2008-01-01

    The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.

  1. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    SciTech Connect

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-17

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. As a result, the active site architecture shows clear evidence of having arisen by convergent evolution.

  2. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    PubMed Central

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-01-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

  3. Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

    PubMed

    Li, Dianfan; Stansfeld, Phillip J; Sansom, Mark S P; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J; Marvin Seibert, M; Caffrey, Martin

    2015-12-17

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

  4. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    NASA Astrophysics Data System (ADS)

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

  5. The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi.

    PubMed Central

    Docampo, R; Pignataro, O P

    1991-01-01

    Using [32P]Pi and [3H]inositol as precursors, we have detected the presence of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and their derivatives inositol phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate respectively, in Trypanosoma cruzi epimastigotes. Using digitonin-permeabilized cells it was possible to detect a stimulation in the formation of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate as well as an increased generation of diacylglycerol in the presence of 1 mM-CaCl2. These results are consistent with the operation of a functional inositol phosphate/diacylglycerol pathway in T. cruzi, and constitute the first demonstration of the presence and activation of this pathway in a parasitic protozoan. These results also indicate that this pathway is conserved during evolution from lower to higher eukaryotic organisms. Images Fig. 1. PMID:2025225

  6. Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

    PubMed

    Li, Dianfan; Stansfeld, Phillip J; Sansom, Mark S P; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J; Marvin Seibert, M; Caffrey, Martin

    2015-01-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

  7. Diacylglycerol kinase as a possible therapeutic target for neuronal diseases.

    PubMed

    Shirai, Yasuhito; Saito, Naoaki

    2014-04-07

    Diacylglycerol kinase (DGK) is a lipid kinase converting diacylglycerol to phosphatidic acid, and regulates many enzymes including protein kinase C, phosphatidylinositol 4-phosphate 5-kinase, and mTOR. To date, ten mammalian DGK subtypes have been cloned and divided into five groups, and they show subtype-specific tissue distribution. Therefore, each DGK subtype is thought to be involved in respective cellular responses by regulating balance of the two lipid messengers, diacylglycerol and phosphatidic acid. Indeed, the recent researches using DGK knockout mice have clearly demonstrated the importance of DGK in the immune system and its pathophysiological roles in heart and insulin resistance in diabetes. Especially, most subtypes show high expression in brain with subtype specific regional distribution, suggesting that each subtype has important and unique functions in brain. Recently, neuronal functions of some DGK subtypes have accumulated. Here, we introduce DGKs with their structural motifs, summarize the enzymatic properties and neuronal functions, and discuss the possibility of DGKs as a therapeutic target of the neuronal diseases.

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

    PubMed

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

    2016-09-01

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

  9. Characterization of the Mouse and Human Monoacylglycerol O-Acyltransferase 1 (Mogat1) Promoter in Human Kidney Proximal Tubule and Rat Liver Cells

    PubMed Central

    Sankella, Shireesha; Garg, Abhimanyu; Agarwal, Anil K.

    2016-01-01

    Monoacylglycerol acyltransferase 1 (Mogat1) catalyzes the conversion of monoacylglycerols (MAG) to diacylglycerols (DAG), the precursor of several physiologically important lipids such as phosphatidylcholine, phosphatidylethanolamine and triacylglycerol (TAG). Expression of Mogat1 is tissue restricted and it is highly expressed in the kidney, stomach and adipose tissue but minimally in the normal adult liver. To understand the transcriptional regulation of Mogat1, we characterized the mouse and human Mogat1 promoters in human kidney proximal tubule-2 (HK-2) cells. In-silico analysis revealed several peroxisome proliferator response element (PPRE) binding sites in the promoters of both human and mouse Mogat1. These sites responded to all three peroxisome proliferator activated receptor (PPAR) isoforms such that their respective agonist or antagonist activated or inhibited the expression of Mogat1. PPRE site mutagenesis revealed that sites located at -592 and -2518 are very effective in decreasing luciferase reporter gene activity. Chromatin immunoprecipitation (ChIP) assay using PPARα antibody further confirmed the occupancy of these sites by PPARα. While these assays revealed the core promoter elements necessary for Mogat1 expression, there are additional elements required to regulate its tissue specific expression. Chromosome conformation capture (3C) assay revealed additional cis-elements located ~10–15 kb upstream which interact with the core promoter. These chromosomal regions are responsive to both PPARα agonist and antagonist. PMID:27611931

  10. Characterization of the Mouse and Human Monoacylglycerol O-Acyltransferase 1 (Mogat1) Promoter in Human Kidney Proximal Tubule and Rat Liver Cells.

    PubMed

    Sankella, Shireesha; Garg, Abhimanyu; Agarwal, Anil K

    2016-01-01

    Monoacylglycerol acyltransferase 1 (Mogat1) catalyzes the conversion of monoacylglycerols (MAG) to diacylglycerols (DAG), the precursor of several physiologically important lipids such as phosphatidylcholine, phosphatidylethanolamine and triacylglycerol (TAG). Expression of Mogat1 is tissue restricted and it is highly expressed in the kidney, stomach and adipose tissue but minimally in the normal adult liver. To understand the transcriptional regulation of Mogat1, we characterized the mouse and human Mogat1 promoters in human kidney proximal tubule-2 (HK-2) cells. In-silico analysis revealed several peroxisome proliferator response element (PPRE) binding sites in the promoters of both human and mouse Mogat1. These sites responded to all three peroxisome proliferator activated receptor (PPAR) isoforms such that their respective agonist or antagonist activated or inhibited the expression of Mogat1. PPRE site mutagenesis revealed that sites located at -592 and -2518 are very effective in decreasing luciferase reporter gene activity. Chromatin immunoprecipitation (ChIP) assay using PPARα antibody further confirmed the occupancy of these sites by PPARα. While these assays revealed the core promoter elements necessary for Mogat1 expression, there are additional elements required to regulate its tissue specific expression. Chromosome conformation capture (3C) assay revealed additional cis-elements located ~10-15 kb upstream which interact with the core promoter. These chromosomal regions are responsive to both PPARα agonist and antagonist. PMID:27611931

  11. 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

  12. 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

  13. 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

  14. Characterisation of two alcohol acyltransferases from kiwifruit (Actinidia spp.) reveals distinct substrate preferences.

    PubMed

    Günther, Catrin S; Chervin, Christian; Marsh, Ken B; Newcomb, Richard D; Souleyre, Edwige J F

    2011-06-01

    Volatile esters are key compounds of kiwifruit flavour and are formed by alcohol acyltransferases that belong to the BAHD acyltransferase superfamily. Quantitative RT-PCR was used to screen kiwifruit-derived expressed sequence tags with proposed acyltransferase function in order to select ripening-specific sequences and test their involvement in alcohol acylation. The screening criterion was for at least 10-fold increased transcript accumulation in ripe compared with unripe kiwifruit and in response to ethylene. Recombinant expression in yeast revealed alcohol acyltransferase activity for Actinidia-derived AT1, AT16 and the phylogenetically distinct AT9, using various alcohol and acyl-CoA substrates. Functional characterisation of AT16 and AT9 demonstrated striking differences in their substrate preferences and apparent catalytic efficiencies (V'(max)K(m)(-1)). Thus revealing benzoyl-CoA:alcohol O-acyltransferase activity for AT16 and acetyl-CoA:alcohol O-acyltransferase activity for AT9. Both kiwifruit-derived enzymes displayed higher reaction rates with butanol compared with ethanol, even though ethanol is the main alcohol in ripe fruit. Since ethyl acetate and ethyl benzoate are major esters in ripe kiwifruit, we suggest that fruit characteristic volatile profiles result from a combination of substrate availability and specificity of individual alcohol acyltransferases.

  15. Inhibitors of diacylglycerol lipases in neurodegenerative and metabolic disorders.

    PubMed

    Janssen, Freek J; van der Stelt, Mario

    2016-08-15

    2-Arachidonoylglycerol (2-AG) is an endocannabinoid that activates the cannabinoid receptors type 1 and 2. It also serves as an important lipid precursor for the eicosanoid signaling pathway. Consequently, 2-AG is involved in many physiological functions, including anxiety, food intake, inflammation, memory, pain sensation and neurotransmission. Diacylglycerol lipases (DAGLs) are the main biosynthetic enzymes for 2-AG and their role in several pathophysiological conditions is currently under investigation. In this Digest we review all DAGL inhibitors reported to date and their effects in preclinical models of neurodegeneration and metabolic disorders. PMID:27394666

  16. The sound velocity measurement in diacylglycerol oil under high pressure

    NASA Astrophysics Data System (ADS)

    Rostocki, A. J.; Malanowski, A.; Tarakowski, R.; Szlachta, K.; Kiełczyński, P.; Szalewski, M.; Balcerzak, A.; Ptasznik, S.

    2013-03-01

    In this article, the influence of high pressure on sound velocity at 293 K has been presented. The investigated diacylglycerol oil (DAG - [D82T18]AG) was composed of 82% DAGs and 18% triacylglycerols. The variation of sound velocity with hydrostatic pressure for DAG was evaluated up to 400 MPa. The phase transformation in DAG has been observed as a discontinuity of the dependence of sound velocity on pressure. The sound velocity during the phase transition has shown distinct increment. Also the volume changes have been measured. It has shown the rapid drop of the volume at the phase transformation pressure due to the possible crystallization of DAG oil.

  17. Inhibitors of diacylglycerol lipases in neurodegenerative and metabolic disorders.

    PubMed

    Janssen, Freek J; van der Stelt, Mario

    2016-08-15

    2-Arachidonoylglycerol (2-AG) is an endocannabinoid that activates the cannabinoid receptors type 1 and 2. It also serves as an important lipid precursor for the eicosanoid signaling pathway. Consequently, 2-AG is involved in many physiological functions, including anxiety, food intake, inflammation, memory, pain sensation and neurotransmission. Diacylglycerol lipases (DAGLs) are the main biosynthetic enzymes for 2-AG and their role in several pathophysiological conditions is currently under investigation. In this Digest we review all DAGL inhibitors reported to date and their effects in preclinical models of neurodegeneration and metabolic disorders.

  18. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    DOE PAGES

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; et al

    2015-12-17

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternarymore » structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. As a result, the active site architecture shows clear evidence of having arisen by convergent evolution.« less

  19. Molecular Pathways: Targeting Diacylglycerol Kinase Alpha in Cancer.

    PubMed

    Purow, Benjamin

    2015-11-15

    Lipid kinases have largely been neglected as targets in cancer, and an increasing number of reports suggest diacylglycerol kinase alpha (DGKα) may be one with promising therapeutic potential. DGKα is one of 10 DGK family members that convert diacylglycerol (DAG) to phosphatidic acid (PA), and both DAG and PA are critical lipid second messengers in the plasma membrane. A host of important oncogenic proteins and pathways affect cancer cells in part through DGKα, including the c-Met and VEGF receptors. Others partially mediate the effects of DGKα inhibition in cancer, such as mTOR and HIF-1α. DGKα inhibition can directly impair cancer cell viability, inhibits angiogenesis, and notably may also boost T-cell activation and enhance cancer immunotherapies. Although two structurally similar inhibitors of DGKα were established decades ago, they have seen minimal in vivo usage, and it is unlikely that either of these older DGKα inhibitors will have utility for cancer. An abandoned compound that also inhibits serotonin receptors may have more translational potential as a DGKα inhibitor, but more potent and specific DGKα inhibitors are sorely needed. Other DGK family members may also provide therapeutic targets in cancer, but require further investigation.

  20. Relative oxidative stability of diacylglycerol and triacylglycerol oils.

    PubMed

    Qi, Jin F; Wang, Xiang Y; Shin, Jung-Ah; Lee, Young-Hwa; Jang, Young-Seok; Lee, Jeung Hee; Hong, Soon-Taek; Lee, Ki-Teak

    2015-03-01

    To compare the oxidative stability between diacylglycerol (DAG) oil and conventional triacylglycerol (TAG) oil (that is, soybean oil), the prepared stripped diacylglycerol oil (SDO) and soybean oil (SSBO) were stored at 60 °C in the dark for 144 h. During storage peroxide values (POVs), contents of aldehydes, unsaturated fatty acids were measured to evaluate the oxidative stabilities of the 2 oils. The results showed the content of C18:2, C18:3, and total unsaturated fatty acid decreased faster in DAG oil than in soybean oil, whereas the decreased rate of C18:1 was similar in 2 oils. Also, both rate constants (K1 and K2) obtained from POV (K1 ) and total aldehydes (K2 ) indicated that DAG oil (K1 = 3.22 mmol/mol FA h(-1) , K2 = 0.023 h(-1)) was oxidized more rapidly than soybean oil (K1 = 2.56 mmol/mol FA h(-1) , K2 = 0.021 h(-1)), which was mainly due to the difference of acylglycerol composition of the 2 oils along with higher C18:3 (9.6%) in SDO than SSBO (5.7%). It is concluded that DAG was more easily oxidized than soybean oil at 60 °C in the dark for 144 h.

  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. Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis.

    PubMed

    Farese, R V; Konda, T S; Davis, J S; Standaert, M L; Pollet, R J; Cooper, D R

    1987-05-01

    The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.

  3. A bifunctional glycosyltransferase from Agrobacterium tumefaciens synthesizes monoglucosyl and glucuronosyl diacylglycerol under phosphate deprivation.

    PubMed

    Semeniuk, Adrian; Sohlenkamp, Christian; Duda, Katarzyna; Hölzl, Georg

    2014-04-01

    Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

  4. An acyltransferase catalyzing the formation of diacylglucose is a serine carboxypeptidase-like protein

    PubMed Central

    Li, Alice X.; Steffens, John C.

    2000-01-01

    1-O-β-acyl acetals serve as activated donors in group transfer reactions involved in plant natural product biosynthesis and hormone metabolism. However, the acyltransferases that mediate transacylation from 1-O-β-acyl acetals have not been identified. We report the identification of a cDNA encoding a 1-O-β-acylglucose-dependent acyltransferase functioning in glucose polyester biosynthesis by Lycopersicon pennellii. The acyltransferase cDNA encodes a serine carboxypeptidase-like protein, with a conserved Ser-His-Asp catalytic triad. Expression of the acyltransferase cDNA in Saccharomyces cerevisiae conferred the ability to disproportionate 1-O-β-acylglucose to diacylglucose. The disproportionation reaction is regiospecific, catalyzing the conversion of two equivalents of 1-O-β-acylglucose to 1,2-di-O-acylglucose and glucose. Diisopropyl fluorophosphate, a transition-state analog inhibitor of serine carboxypeptidases, inhibited acyltransferase activity and covalently labeled the purified acyltransferase, suggesting the involvement of an active serine in the mechanism of the transacylation. The acyltransferase exhibits no carboxypeptidase activity; conversely, the serine carboxypeptidases we have tested show no ability to transacylate using 1-O-acyl-β-glucoses. This acyltransferase may represent one member of a broader class of enzymes recruited from proteases that have adapted a common catalytic mechanism of catabolism and modified it to accommodate a wide range of group transfer reactions used in biosynthetic reactions of secondary metabolism. The abundance of serine carboxypeptidase-like proteins in plants suggests that this motif has been used widely for metabolic functions. PMID:10829071

  5. Functional characterization of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 10/glycerol-3-phosphate acyltransferase isoform 3

    PubMed Central

    Sukumaran, Suja; Barnes, Robert I; Garg, Abhimanyu; Agarwal, Anil K

    2016-01-01

    Synthesis of phospholipids can occur de novo or via remodeling of the existing phospholipids. Synthesis of triglycerides, a form of energy storage in cells, is an end product of these pathways. Several 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs) acylate lysophosphatidic acid (LPA) at the sn-2 (carbon 2) position to produce phosphatidic acid (PA). These enzymes are involved in phospholipids and triglyceride synthesis through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. We cloned a cDNA predicted to be an AGPAT isoform (AGPAT10). This cDNA has been recently identified as glycerol-3-phosphate-O-acyltransferase isoform 3 (GPAT3). When this AGPAT10/GPAT3 cDNA was expressed in Chinese Hamster ovary cells, the protein product localizes to the endoplasmic reticulum. In vitro enzymatic activity using lysates of human embryonic kidney-293 cells infected with recombinant AGPAT10/GPAT3 adenovirus show that the protein has a robust AGPAT activity with an apparent Vmax of 2 nmol/min per mg protein, but lacks GPAT enzymatic activity. This AGPAT has similar substrate specificities for LPA and acyl-CoA as shown for another known isoform, AGPAT2. We further show that when overexpressed in human Huh-7 cells depleted of endogenous AGPAT activity by sh-RNA-AGPAT2-lentivirus, the protein again demonstrates AGPAT activity. These observations strongly suggest that the cDNA previously identified as GPAT3 has AGPAT activity and thus we prefer to identify this clone as AGPAT10 as well. PMID:19318427

  6. Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries. Evidence that accumulation of diacylglycerol is not a prerequisite for contraction.

    PubMed

    Ohanian, J; Ollerenshaw, J; Collins, P; Heagerty, A

    1990-05-25

    The production of total amounts of 1,2-diacylglycerol as well as those specifically derived from inositol lipid hydrolysis was studied in intact rat resistance arteries stimulated with either noradrenaline, vasopressin, or angiotensin II at 20 s when the onset of contraction would be nearing its maximum, and at 5 min during the sustained phase of contraction. Total amounts of 1,2-diacylglycerol were not altered by any agonist at 20 s, or at 5 min. However, arachidonate-containing species of 1,2-diacylglycerol were differentially influenced being increased at 5 min by noradrenaline, and decreased at 20 s and 5 min by vasopressin. Only angiotensin II produced substantial increases in this class of 1,2-diacylglycerol at both time points. In order to investigate the fate of this second messenger total and inositol lipid derived phosphatidic acids were then measured at both 20 s and 5 min. Noradrenaline induced a rise in both total and arachidonate-containing phosphatidic acid at both times as did vasopressin. Only small increases were induced by angiotensin II at 20 s. These data demonstrate that the accumulation of 1,2-diacylglycerol generated from inositol lipid breakdown is only observed with activation by angiotensin II. Other agonists produced phosphatidic acids with time and the rate of generation of these lipids is agonist-specific. Thus phosphatidic acid may play a more prominent role during the sustained phase of contraction than previously anticipated.

  7. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

    PubMed Central

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins. PMID:27630986

  8. Photoswitchable diacylglycerols enable optical control of protein kinase C.

    PubMed

    Frank, James Allen; Yushchenko, Dmytro A; Hodson, David J; Lipstein, Noa; Nagpal, Jatin; Rutter, Guy A; Rhee, Jeong-Seop; Gottschalk, Alexander; Brose, Nils; Schultz, Carsten; Trauner, Dirk

    2016-09-01

    Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling. PMID:27454932

  9. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity

    PubMed Central

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins.

  10. Diacylglycerol Kinases in the Coordination of Synaptic Plasticity.

    PubMed

    Lee, Dongwon; Kim, Eunjoon; Tanaka-Yamamoto, Keiko

    2016-01-01

    Synaptic plasticity is activity-dependent modification of the efficacy of synaptic transmission. Although, detailed mechanisms underlying synaptic plasticity are diverse and vary at different types of synapses, diacylglycerol (DAG)-associated signaling has been considered as an important regulator of many forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Recent evidences indicate that DAG kinases (DGKs), which phosphorylate DAG to phosphatidic acid to terminate DAG signaling, are important regulators of LTP and LTD, as supported by the results from mice lacking specific DGK isoforms. This review will summarize these studies and discuss how specific DGK isoforms distinctly regulate different forms of synaptic plasticity at pre- and postsynaptic sites. In addition, we propose a general role of DGKs as coordinators of synaptic plasticity that make local synaptic environments more permissive for synaptic plasticity by regulating DAG concentration and interacting with other synaptic proteins. PMID:27630986

  11. Protein purification and cloning of diacylglycerol lipase from rat brain.

    PubMed

    Aso, Chizu; Araki, Mari; Ohshima, Noriyasu; Tatei, Kazuaki; Hirano, Tohko; Obinata, Hideru; Kishi, Mikiko; Kishimoto, Koji; Konishi, Akimitsu; Goto, Fumio; Sugimoto, Hiroyuki; Izumi, Takashi

    2016-06-01

    Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.

  12. Viscosity and compressibility of diacylglycerol under high pressure

    NASA Astrophysics Data System (ADS)

    Malanowski, Aleksander; Rostocki, A. J.; Kiełczyński, P.; Szalewski, M.; Balcerzak, A.; Kościesza, R.; Tarakowski, R.; Ptasznik, S.; Siegoczyński, R. M.

    2013-03-01

    The influence of high pressure on viscosity and compressibility of diacylglycerol (DAG) oil has been presented in this paper. The investigated DAG oil was composed of 82% of DAGs and 18% TAGs (triacylglycerols). The dynamic viscosity of DAG was investigated as a function of the pressure up to 400 MPa. The viscosity was measured by means of the surface acoustic wave method, where the acoustic waveguides were used as sensing elements. As the pressure was rising, the larger ultrasonic wave attenuation was observed, whereas amplitude decreased with the liquid viscosity augmentation. Measured changes of physical properties were most significant in the pressure range near the phase transition. Deeper understanding of DAG viscosity and compressibility changes versus pressure could shed more light on thermodynamic properties of edible oils.

  13. A novel mechanism for acetylcholine to generate diacylglycerol in brain

    SciTech Connect

    Qian, Z.; Drewes, L.R. )

    1990-03-05

    The classical scheme involving inositol phospholipid breakdown by phospholipase C as the sole source of diacylglycerol (DAG) has recently been challenged by evidence that phosphatidylcholine (PC) is an alternative source. In synaptic membranes of canine cerebral cortex, cholinergic agonists caused rapid accumulation of ({sup 3}H)phosphatidic acid (PA) from ({sup 3}H)PC within 15 s, whereas (3H)DAG formation showed a transient lag period before becoming elevated and then exceeding the amount of ({sup 3}H)PA. Additional evidence shows that DAG is produced from PC by the action of phospholipase D to yield PA, which is further dephosphorylated to DAG by PA phosphatase. Our results indicate that this muscarinic acetylcholine receptor-regulated PC phospholipase D-PA phosphatase pathway may be a novel mechanism in cell signal transduction processes for activation of protein kinase C in brain.

  14. Diacylglycerol Signaling Pathway in Pancreatic β-Cells: An Essential Role of Diacylglycerol Kinase in the Regulation of Insulin Secretion.

    PubMed

    Kaneko, Yukiko K; Ishikawa, Tomohisa

    2015-01-01

    Diacylglycerol (DAG) is a lipid signal messenger and plays a physiological role in β-cells. Since defective glucose homeostasis increases de novo DAG synthesis, DAG may also contribute to β-cell dysfunction in type 2 diabetes. Although the primary function of DAG is to activate protein kinase C (PKC), the role of PKC in insulin secretion is controversial: PKC has been reported to act as both a positive and negative regulator of insulin secretion. In addition to the PKC pathway, DAG has also been shown to mediate other pathways such as the Munc-13-dependent pathway in β-cells. The intracellular levels of DAG are strictly regulated by diacylglycerol kinase (DGK); however, the role of DGK in β-cells and their involvement in β-cell failure in type 2 diabetes remain to be fully elucidated. We have recently reported the roles of type I DGK, DGKα and γ, in insulin secretion from β-cells. DGKα and γ were activated by glucose or high K(+) stimulation in β-cells, and the inhibition of the DGKs by a type I DGK inhibitor or by knockdown with small interfering RNA (siRNA) decreased insulin secretion. Thus, DGKα and γ are suggested to be activated in response to elevated [Ca(2+)]i in β-cells and to act as positive regulators of insulin secretion. In this article, we review the current understanding of the roles of DAG and DGK in β-cell function and their involvement in the development of β-cell dysfunction in type 2 diabetes.

  15. Gene Expression in Plant Lipid Metabolism in Arabidopsis Seedlings

    PubMed Central

    Hsiao, An-Shan; Haslam, Richard P.; Michaelson, Louise V.; Liao, Pan; Napier, Johnathan A.; Chye, Mee-Len

    2014-01-01

    Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG) synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3), DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS) was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6) in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX) lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis. PMID:25264899

  16. Ghrelin O-acyltransferase (GOAT) and energy metabolism.

    PubMed

    Li, Ziru; Mulholland, Michael; Zhang, Weizhen

    2016-03-01

    Ghrelin O-acyltransferase (GOAT), a member of MBOATs family, is essential for octanoylation of ghrelin, which is required for active ghrelin to bind with and activate its receptor. GOAT is expressed mainly in the stomach, pancreas and hypothalamus. Levels of GOAT are altered by energy status. GOAT contains 11 transmembrane helices and one reentrant loop. Its invariant residue His-338 and conserved Asn-307 are located in the endoplasmic reticulum lumen and cytosol respectively. GOAT contributes to the regulation of food intake and energy expenditure, as well as glucose and lipids homeostasis. Deletion of GOAT blocks the acylation of ghrelin leading to subsequent impairment in energy homeostasis and survival when mice are challenged with high energy diet or severe caloric restriction. GO-CoA-Tat, a peptide GOAT inhibitor, attenuates acyl-ghrelin production and prevents weight gain induced by a medium-chain triglycerides-rich high fat diet. Further, GO-CoA-Tat increases glucose- induced insulin secretion. Overall, inhibition of GOAT is a novel strategy for treatment of obesity and related metabolic disorders. PMID:26732975

  17. Life and death among plant lysophosphatidic acid acyltransferases.

    PubMed

    Maisonneuve, Sylvie; Guyot, Romain; Roscoe, Thomas

    2010-07-01

    The tetraploid Brassica napus possesses several seed-expressed microsomal lysophosphatidic acid acyltransferases (LPAAT ) including BAT1.5, which has been retained after genome duplication as a consequence of a subfunctionalisation of the gene encoding the ubiquitously expressed Kennedy pathway enzyme BAT1.13. Next, cDNA BAT1.3, encoding a LPAAT was subsequently isolated from an embryo library. The rapeseed LPAAT encoded by BAT1.3 is orthologous to the Arabidopsis thaliana At1g51260 gene product possibly associated with tapetum development and male fertility. However, BAT1.3 expression is predominant during the mid stages of embryo development in seeds of Brassica napus. Functional characterisation of BAT1.3 provides further support for a hypothesis of gene dosage sensitivity of LPAATs as does an analysis of the chromosomal localisation of LPAAT genes in Arabidopsis thaliana. The pattern of retention or loss of LPAAT genes after polyploidisation or segmental duplication is consistent with a model of balanced gene drive.

  18. Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase.

    PubMed

    Ferri, S R; Toguri, T

    1997-01-15

    The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

  19. 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

  20. 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

  1. 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

  2. Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

    SciTech Connect

    Faellman, M.; Stendahl, O.; Andersson, T. )

    1989-03-01

    Human neutrophils stimulated with a phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 minutes. In contrast, 4-{alpha}-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.

  3. Diacylglycerol Kinase-ε: Properties and Biological Roles

    PubMed Central

    Epand, Richard M.; So, Vincent; Jennings, William; Khadka, Bijendra; Gupta, Radhey S.; Lemaire, Mathieu

    2016-01-01

    In mammals there are at least 10 isoforms of diacylglycerol kinases (DGK). All catalyze the phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PA). Among DGK isoforms, DGKε has several unique features. It is the only DGK isoform with specificity for a particular species of DAG, i.e., 1-stearoyl-2-arachidonoyl glycerol. The smallest of all known DGK isoforms, DGKε, is also the only DGK devoid of a regulatory domain. DGKε is the only DGK isoform that has a hydrophobic segment that is predicted to form a transmembrane helix. As the only membrane-bound, constitutively active DGK isoform with exquisite specificity for particular molecular species of DAG, the functional overlap between DGKε and other DGKs is predicted to be minimal. DGKε exhibits specificity for DAG containing the same acyl chains as those found in the lipid intermediates of the phosphatidylinositol-cycle. It has also been shown that DGKε affects the acyl chain composition of phosphatidylinositol in whole cells. It is thus likely that DGKε is responsible for catalyzing one step in the phosphatidylinositol-cycle. Steps of this cycle take place in both the plasma membrane and the endoplasmic reticulum membrane. DGKε is likely present in both of these membranes. DGKε is the only DGK isoform that is associated with a human disease. Indeed, recessive loss-of-function mutations in DGKε cause atypical hemolytic-uremic syndrome (aHUS). This condition is characterized by thrombosis in the small vessels of the kidney. It causes acute renal insufficiency in infancy and most patients develop end-stage renal failure before adulthood. Disease pathophysiology is poorly understood and there is no therapy. There are also data suggesting that DGKε may play a role in epilepsy and Huntington disease. Thus, DGKε has many unique molecular and biochemical properties when compared to all other DGK isoforms. DGKε homologs also contain a number of conserved sequence features that are distinctive

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

    SciTech Connect

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

    2015-05-08

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

  5. Insulin activates glycerol-3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator. Apparent involvement of Gi alpha and activation of a phospholipase C.

    PubMed

    Vila, M C; Milligan, G; Standaert, M L; Farese, R V

    1990-09-18

    We studied the mechanism whereby insulin activates de novo phosphatidic acid synthesis in BC3H-1 myocytes. Insulin rapidly activated glycerol-3-phosphate acyltransferase (G3PAT) in intact and cell-free preparations of myocytes in a dose-related manner. The apparent Km of the enzyme was decreased by treatment with insulin, whereas the Vmax was unaffected. No activation was found by ACTH, insulin-like growth factor-I, angiotensin II, or phenylephrine, but epidermal growth factor, which, like insulin, is known to activate de novo phosphatidic acid synthesis in intact myocytes, also stimulated G3PAT activity. In homogenates or membrane fractions, the effect of insulin on G3PAT was fully mimicked by nonspecific or phosphatidylinositol (PI)-specific phospholipase C (PLC). An antiserum raised against PI-glycan-PLC completely blocked the effect of insulin on G3PAT. Although the above findings suggested involvement of a PLC in insulin-induced activation of G3PAT, neither diacylglycerol nor protein kinase C activation appeared to be involved. On the other hand, insulin stimulated the release of a cytosolic factor, which activated membrane-associated G3PAT. This cytosolic factor had a molecular weight of less than 5K as determined by Sephadex G-25 chromatography. NaF, a phosphatase inhibitor, blocked the activation of G3PAT by insulin, suggesting involvement of a phosphatase. Insulin-induced activation of G3PAT was also blocked by pretreatment of intact myocytes with pertussis toxin and by prior addition, to homogenates, of an antiserum that recognizes the C-terminal decapeptide of Gi alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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

  7. Cytosolic rat brain synapsin I is a diacylglycerol kinase.

    PubMed Central

    Kahn, D W; Besterman, J M

    1991-01-01

    The phosphorylation of diacylglycerol (DG), a reaction catalyzed by DG kinase, may be critical in the termination of effector-induced signals mediated by protein kinase C. Synapsin I is a principal target of intracellular protein kinases and is thought to be involved in the release of neurotransmitter from axon terminals. We present several lines of evidence which indicate that rat brain synapsin, in addition to this role, may function as a DG kinase. Purified rat brain DG kinase was digested with trypsin, which produced three major fragments whose sequence was identical to three regions in synapsin I. Using a rabbit anti-synapsin polyclonal antiserum, the elution profile of synapsin immunoreactivity coincided exactly with that of DG kinase activity in column fractions from the final step in the DG kinase purification procedure. As is the case with synapsin, the purified enzyme was a strongly basic protein with an isoelectric point greater than 10.0. Finally, incubating the DG kinase with highly purified bacterial collagenase, an enzyme that partially degrades the proline- and glycine-rich synapsin, resulted in the simultaneous loss of DG kinase activity and synapsin immunoreactivity. We conclude that cytosolic rat brain synapsin is capable of functioning as a DG kinase. Images PMID:1648730

  8. Biosynthesis of alkyl lysophosphatidic acid by diacylglycerol kinases.

    PubMed

    Gellett, Amanda M; Kharel, Yugesh; Sunkara, Manjula; Morris, Andrew J; Lynch, Kevin R

    2012-06-15

    Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.

  9. A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

    PubMed Central

    Mizushina, Yoshiyuki; Xu, Xianai; Asahara, Hitomi; Takeuchi, Ryo; Oshige, Masahiko; Shimazaki, Noriko; Takemura, Masaharu; Yamaguchi, Toyofumi; Kuroda, Kazufumi; Linn, Stuart; Yoshida, Hiromi; Koiwai, Osamu; Saneyoshi, Mineo; Sugawara, Fumio; Sakaguchi, Kengo

    2003-01-01

    Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon. PMID:12435270

  10. Diacylglycerol lipase disinhibits VTA dopamine neurons during chronic nicotine exposure.

    PubMed

    Buczynski, Matthew W; Herman, Melissa A; Hsu, Ku-Lung; Natividad, Luis A; Irimia, Cristina; Polis, Ilham Y; Pugh, Holly; Chang, Jae Won; Niphakis, Micah J; Cravatt, Benjamin F; Roberto, Marisa; Parsons, Loren H

    2016-01-26

    Chronic nicotine exposure (CNE) alters synaptic transmission in the ventral tegmental area (VTA) in a manner that enhances dopaminergic signaling and promotes nicotine use. The present experiments identify a correlation between enhanced production of the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and diminished release of the inhibitory neurotransmitter GABA in the VTA following CNE. To study the functional role of on-demand 2-AG signaling in GABAergic synapses, we used 1,2,3-triazole urea compounds to selectively inhibit 2-AG biosynthesis by diacylglycerol lipase (DAGL). The potency and selectivity of these inhibitors were established in rats in vitro (rat brain proteome), ex vivo (brain slices), and in vivo (intracerebroventricular administration) using activity-based protein profiling and targeted metabolomics analyses. Inhibition of DAGL (2-AG biosynthesis) rescues nicotine-induced VTA GABA signaling following CNE. Conversely, enhancement of 2-AG signaling in naïve rats by inhibiting 2-AG degradation recapitulates the loss of nicotine-induced GABA signaling evident following CNE. DAGL inhibition reduces nicotine self-administration without disrupting operant responding for a nondrug reinforcer or motor activity. Collectively, these findings provide a detailed characterization of selective inhibitors of rat brain DAGL and demonstrate that excessive 2-AG signaling contributes to a loss of inhibitory GABAergic constraint of VTA excitability following CNE.

  11. Diacylglycerol kinase activity in brain cytosol and microsomes

    SciTech Connect

    Kelleher, J.A.; Sun, G.Y.

    1986-05-01

    The ATP-dependent diacylglycerol (DG) kinase phosphorylated DG to form phosphatidic acids (PA). This enzymic conversion is particularly important in the receptor-mediated polyphosphoinositide metabolism. Controlling the DG level in synaptic membranes can also modulate the protein kinase activity within the cell. Using /sup 32/P-ATP, MgCl/sub 2/, NaF and heat treated membranes as substrate, DG-kinase activity was found in both cytosolic and microsomal fractions. Similarities in properties between the two kinase activities were noted. For example, activities in both fractions were stimulated by deoxycholate, and were inhibited by dibucaine and propranol. These results suggest that the microsomal and cytosolic DG-kinase(s) may belong to the same enzyme and that some intracellular factors may be responsible for regulation of the enzyme for interaction with membrane substrate. One of the factors tested was free fatty acid (FFA) which appeared to promote translocation of the cytosolic enzyme to the microsomes. Another factor for regulation is the availability of DG, which is formed via the poly-PI phosphodiesterase in synaptosomes and PA-phosphohydrolase in the microsomes (for de novo biosynthesis of phospholipids). Possible physiological significance of these regulatory mechanisms will be addressed.

  12. CDP-diacylglycerol synthase activity in Clostridium perfingens

    SciTech Connect

    Carmen, G.M.; Zaniewski, R.L.; Cousminer, J.J.

    1982-01-01

    CTP: phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimium between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition on the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C. (Refs. 24).

  13. Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast

    PubMed Central

    Ganesan, Suriakarthiga; Shabits, Brittney N.; Zaremberg, Vanina

    2015-01-01

    Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed. PMID:27081314

  14. Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

    SciTech Connect

    Kelley, M.J.; Carman, G.M.

    1987-10-25

    The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.

  15. Diacylglycerol kinase α exacerbates cardiac injury after ischemia/reperfusion.

    PubMed

    Sasaki, Toshiki; Shishido, Tetsuro; Kadowaki, Shinpei; Kitahara, Tatsuro; Suzuki, Satoshi; Katoh, Shigehiko; Funayama, Akira; Netsu, Shunsuke; Watanabe, Tetsu; Goto, Kaoru; Takeishi, Yasuchika; Kubota, Isao

    2014-01-01

    Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal for the preservation of myocardial function. However, reperfusion itself causes additional myocardium injuries. Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade has been implicated in the cardioprotective effects occurring after ischemia/reperfusion (I/R). DAG kinase (DGK) controls cellular DAG levels by converting DAG to phosphatidic acid, and may act as an endogenous regulator of DAG-PKC signaling. In the present study, we examined the functional role of DGKα in cardiac injury after I/R in in vivo mouse hearts. We generated transgenic mice with cardiac-specific overexpression of DGKα (DGKα-TG). The left anterior descending coronary artery was transiently occluded for 20 min and reperfused for 24 h in DGKα-TG mice and wild-type littermate (WT) mice. The levels of phosphorylation activity of PKCε, extracellular-signal regulated kinase (ERK) 1/2, and p70 ribosomal S6 kinase (p70S6K) were increased after I/R in WT mouse hearts. However, in DGKα-TG mice, activation of PKCε, ERK1/2, and p70S6K was attenuated compared to WT mice. After 24 h, Evans blue/triphenyltetrazolium chloride double staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed that DGKα-TG mice had significantly larger myocardial infarctions and larger numbers of TUNEL-positive cardiomyocytes than WT mice. Echocardiography and cardiac catheterization revealed that left ventricular systolic function was more severely depressed in DGKα-TG mice than in WT mice after I/R. These findings suggest that DGKα exacerbates I/R injury by inhibiting the cardioprotective effects of PKCε, ERK1/2, and p70S6K activation. PMID:23719772

  16. Negative regulation of mTOR activation by diacylglycerol kinases

    PubMed Central

    Gorentla, Balachandra K.; Wan, Chi-Keung

    2011-01-01

    The engagement of TCR induces T-cell activation, which initiates multiple characteristic changes such as increase in cell size, cell division, and the production of cytokines and other effector molecules. The mammalian target of rapamycin (mTOR) regulates protein synthesis, transcription, cell survival, and autophagy. Critical roles of mTOR in T-cell activation and effector/memory differentiation have been revealed using chemical inhibitors or by genetic ablation of mTOR in T cells. However, the connection between mTOR signaling and other signaling cascades downstream of TCR is unclear. We demonstrate that diacylglycerol (DAG) and TCR engagement activate signaling in both mTOR complexes 1 and 2 through the activation of the Ras–mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 (Mek1/2)–extracellular signal–regulated kinase 1/2 (Erk1/2)–activator protein 1 (AP-1), known collectively as the Ras-Mek1/2-Erk1/2-AP-1 pathway. Deficiency of RasGRP1 or inhibition of Mek1/2 activity drastically decreases TCR-induced mTOR activation, whereas constitutively active Ras or Mek1 promotes mTOR activation. Although constitutively active Akt promotes TCR-induced mTOR activation, such activation is attenuated by Mek1/2 inhibition. We demonstrated further that DAG kinases (DGKs) α and ζ, which terminate DAG-mediated signaling, synergistically inhibit TCR-induced mTOR activation by inhibiting the Ras-Mek1/2-Erk/12 pathway. These observations provide novel insights into the regulation of mTOR activation. PMID:21310925

  17. Cytochalasin B augments diacylglycerol levels in stimulated neutrophils

    SciTech Connect

    Honeycutt, P.J.; Niedel, J.

    1986-03-05

    Diacylglycerol (DG) has gained wide acceptance as an important second messenger and intracellular activator of protein kinase C, but few studies have directly measured DG levels in cells or tissues. The authors measured the mass of DG in lipid extracts from normal human neutrophils by quantitative conversion of DG to (/sup 32/P) phosphatidic acid using E. coli DG kinase. The chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) stimulated a transient 30% rise in DG that was maximal at 30 to 45 sec and returned to the basal level of 150 picomoles/10/sup 7/ cells by one min. This initial peak was followed by a slower, more prolonged 30% increase in DG that was maximal at 20 min. Cytochalasin B (CB) augments many biological responses of neutrophils to fMLP, including superoxide production and lysosomal enzyme release. CB alone caused no change in basal DG levels, but in the presence of CB, fMLP stimulated a rapid, large, and persistent DG response. DG levels increased to 290% of basal at 5 min with a t1/2 = 45 sec. The DG response to fMLP was maximal at 5 to 10 ..mu..m CB and 1 ..mu..M fMLP. The DG response to optimal fMLP and CB concentrations was decreased 40% by an fMLP antagonist, and no response was elicited by an inactive fMLP analog and CB. Protein kinase C has been implicated in fMLP-stimulated superoxide production and lysosomal enzyme release. These data are consistent with the hypothesis that CB may effect augmentation of biological responses by increasing DG levels.

  18. Phosphoryl transfer reaction catalyzed by membrane diacylglycerol kinase: a theoretical mechanism study.

    PubMed

    Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Li, Xichen; Chen, Guangju; Jia, Zongchao

    2015-10-14

    Diacylglycerol kinase is an integral membrane protein which catalyzes phosphoryl transfer from ATP to diacylglycerol. As the smallest kinase known, it shares no sequence homology with conventional kinases and possesses a distinct trimer structure. Thus far, its catalytic mechanism remains elusive. Using molecular dynamics and quantum mechanics calculations, we investigated the co-factor and the substrate binding and phosphoryl transfer mechanism. Based on the analysis of density functional theory calculations, we reveal that the phosphorylation reaction of diacylglycerol kinase features the same phosphoryl transfer mechanism as other kinases, despite its unique structural properties. Our results further show that the active site is relatively open and able to accommodate ligands in multiple orientations, suggesting that the optimization of binding orientations and conformational changes would occur prior to actual phosphoryl transfer.

  19. Rapid flip-flop motions of diacylglycerol and ceramide in phospholipid bilayers

    NASA Astrophysics Data System (ADS)

    Ogushi, Fumiko; Ishitsuka, Reiko; Kobayashi, Toshihide; Sugita, Yuji

    2012-01-01

    We have investigated flip-flop motions of diacylglycerol and ceramide in phospholipid bilayers using coarse-grained molecular dynamics simulations. In the simulations, flip-flop motions of diacylglycerol and ceramide in the DAPC membrane are slower than cholesterol. Rates correlate with the number of unsaturated bonds in the membrane phospholipids and hence with fluidity of membranes. These findings qualitatively agree with corresponding experimental data. Statistical analysis of the trajectories suggests that flip-flop can be approximated as a Poisson process. The rate of the transverse movement is influenced by depth of the polar head group in the membrane and extent of interaction with water.

  20. 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

  1. Unusual metal ion catalysis in an acyl-transferase ribozyme.

    PubMed

    Suga, H; Cowan, J A; Szostak, J W

    1998-07-14

    Most studies of the roles of catalytic metal ions in ribozymes have focused on inner-sphere coordination of the divalent metal ions to the substrate or ribozyme. However, divalent metal ions are strongly hydrated in water, and some proteinenzymes, such as Escherichia coli RNase H and exonuclease III, are known to use metal cofactors in their fully hydrated form [Duffy, T. H., and Nowak, T. (1985) Biochemistry 24, 1152-1160; Jou, R., and Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686]. It is therefore important to consider the possibility of outer-sphere coordination of catalytic metal ions in ribozymes. We have used an exchange-inert metal complex, cobalt hexaammine, to show that the catalytic metal ion in an acyl-transferase ribozyme acts through outer-sphere coordination. Our studies provide an example of a fully hydrated Mg2+ ion that plays an essential role in ribozyme catalysis. Kinetic studies of wild-type and mutant ribozymes suggest that a pair of tandem G:U wobble base pairs adjacent to the reactive center constitute the metal-binding site. This result is consistent with recent crystallographic studies [Cate, J. H., and Doudna, J. A. (1996) Structure 4, 1221-1229; Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685; Cate, J. H., Hanna, R. L., and Doudna, J. A. (1997) Nat. Struct. Biol. 4, 553-558] showing that tandem wobble base pairs are good binding sites for metal hexaammines. We propose a model in which the catalytic metal ion is bound in the major groove of the tandem wobble base pairs, is precisely positioned by the ribozyme within the active site, and stabilizes the developing oxyanion in the transition state. Our results may have significant implications for understanding the mechanism of protein synthesis [Noller, H. F., Hoffarth, V., and Zimniak, L. (1992) Science 256, 1416-1419].

  2. Purification and characterization of diacylglycerol lipase from human platelets.

    PubMed

    Moriyama, T; Urade, R; Kito, M

    1999-06-01

    Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a combination of chromatographic and electrophoretic methods. The molecular mass of the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as determined by Immobiline isoelectro-focusing. The enzymatic activity of the partially purified DGL was investigated in the presence of a variety of inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2 inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) enhanced it. In addition, the enzymatic activity was inhibited by two serine blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB). These results suggest that cysteine, serine and histidine residues are required for the enzymatic activity of DGL. DGL was optimally active in the pH range of 7-8 and its activity did not change significantly in the presence of various calcium concentrations, even in the presence of 2 mM EGTA. This indicates that DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a dose-dependent manner with an IC50 (the concentration required for 50% inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2) inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol, 2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most potent inhibitor.

  3. Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.

    PubMed

    Jerga, Agoston; Lu, Ying-Jie; Schujman, Gustavo E; de Mendoza, Diego; Rock, Charles O

    2007-07-27

    Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

  4. Topology and Secondary Structure of the N-terminal Domain of Diacylglycerol Kinase

    SciTech Connect

    Oxenoid, Kirill; Soennichsen, Frank D.; Sanders, Charles R.

    2002-09-28

    Prokaryotic diacylglycerol kinase (DAGK) functions as a homotrimer of 13 kDa subunits, each of which has three transmembrane segments. This enzyme is conditionally essential to some bacteria and serves as a model system for studies of membrane protein biocatalysis, stability, folding, and misfolding. In this work, the detailed topology and secondary structure of DAGKs N-terminus up through the loop

  5. Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown

    SciTech Connect

    Catalioto, R.M.; Ailhaud, G.; Negrel, R. )

    1990-12-31

    Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with ({sup 3}H)glycerol or ({sup 3}H)choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in ({sup 3}H)ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 ({sup 3}H))phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.

  6. 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

  7. Secretagogue-induced diacylglycerol accumulation in isolated pancreatic islets. Mass spectrometric characterization of the fatty acyl content indicates multiple mechanisms of generation

    SciTech Connect

    Wolf, B.A.; Easom, R.A.; Hughes, J.H.; McDaniel, M.L.; Turk, J. )

    1989-05-16

    Diacylglycerol accumulation has been examined in secretagogue-stimulated pancreatic islets with a newly developed negative ion chemical ionization mass spectrometric method. The muscarinic agonist carbachol induces islet accumulation of diacylglycerol rich in arachidonate and stearate, and a parallel accumulation of {sup 3}H-labeled diacylglycerol occurs in carbachol-stimulated islets that had been prelabeled with ({sup 3}H)glycerol. Islets so labeled do not accumulate {sup 3}H-labeled diacylglycerol in response to D-glucose, but D-glucose does induce islet accumulation of diacylglycerol by mass. This material is rich in palmitate and oleate and contains much smaller amounts of arachidonate. Neither secretagogue influences triacylglycerol labeling, and neither induces release of ({sup 3}H)choline or ({sup 3}H)phosphocholine from islets prelabeled with ({sup 3}H)choline. These observations indicate that the diacylglycerol that accumulates in islets in response to carbachol arises from hydrolysis of glycerolipids, probably including phosphoinositides. The bulk of the diacylglycerol which accumulates in response to glucose does not arise from glycerolipid hydrolysis and must therefore reflect de novo synthesis. The endogenous diacylglycerol which accumulates in secretagogue-stimulated islets may participate in insulin secretion because exogenous diacylglycerol induces insulin secretion from islets, and an inhibitor of diacylglycerol metabolism to phosphatidic acid augments glucose-induced insulin secretion.

  8. 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

  9. 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

  10. Diacylglycerol Kinases (DGKs): Novel Targets for Improving T Cell Activity in Cancer

    PubMed Central

    Riese, Matthew J.; Moon, Edmund K.; Johnson, Bryon D.; Albelda, Steven M.

    2016-01-01

    Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the metabolism of diacylglycerol (DAG). Two isoforms of DGK, DGKα, and DGKζ, specifically regulate the pool of DAG that is generated as a second messenger after stimulation of the T cell receptor (TCR). Deletion of either isoform in mouse models results in T cells bearing a hyperresponsive phenotype and enhanced T cell activity against malignancy. Whereas, DGKζ appears to be the dominant isoform in T cells, rationale exists for targeting both isoforms individually or coordinately. Additional work is needed to rigorously identify the molecular changes that result from deletion of DGKs in order to understand how DAG contributes to T cell activation, the effect of DGK inhibition in human T cells, and to rationally develop combined immunotherapeutic strategies that target DGKs. PMID:27800476

  11. Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

    PubMed

    Chibalin, Alexander V; Leng, Ying; Vieira, Elaine; Krook, Anna; Björnholm, Marie; Long, Yun Chau; Kotova, Olga; Zhong, Zhihui; Sakane, Fumio; Steiler, Tatiana; Nylén, Carolina; Wang, Jianjun; Laakso, Markku; Topham, Matthew K; Gilbert, Marc; Wallberg-Henriksson, Harriet; Zierath, Juleen R

    2008-02-01

    Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life. PMID:18267070

  12. Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli.

    PubMed Central

    Dutt, A; Dowhan, W

    1981-01-01

    The synthesis of phosphatidylserine in two gram-positive aerobic bacteria has been partially characterized. We have located a cytidine 5'-diphospho-diacylglycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase) activity in the membrane fraction of Bacillus licheniformis and Bacillus subtilis. The activity was demonstrated to be membrane associated by differential centrifugation, sucrose gradient centrifugation, and detergent solubilization. The direct involvement of cytidine 5'-diphospho-diacylglycerol in the reaction was demonstrated by the conversion of the liponucleotide phosphatidyl moiety to phosphatidylserine. This activity is dependent on divalent metal ion (manganese being optimal) and is stimulated by nonionic detergent and its product phosphatidylserine. Based on studies with various combinations of products and substrates, the reaction appears to follow a sequential BiBi kinetic mechanism. PMID:6267011

  13. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    PubMed Central

    Weigel, Christoph; Veldwijk, Marlon R.; Oakes, Christopher C.; Seibold, Petra; Slynko, Alla; Liesenfeld, David B.; Rabionet, Mariona; Hanke, Sabrina A.; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  14. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.

    PubMed

    Weigel, Christoph; Veldwijk, Marlon R; Oakes, Christopher C; Seibold, Petra; Slynko, Alla; Liesenfeld, David B; Rabionet, Mariona; Hanke, Sabrina A; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-03-11

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy.

  15. Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition.

    PubMed

    Ogasawara, Daisuke; Deng, Hui; Viader, Andreu; Baggelaar, Marc P; Breman, Arjen; den Dulk, Hans; van den Nieuwendijk, Adrianus M C H; van den Nieuwendijk, Adriann M C H; Soethoudt, Marjolein; van der Wel, Tom; Zhou, Juan; Overkleeft, Herman S; Sanchez-Alavez, Manuel; Mori, Simone; Mo, Simone; Nguyen, William; Conti, Bruno; Liu, Xiaojie; Chen, Yao; Liu, Qing-Song; Cravatt, Benjamin F; van der Stelt, Mario

    2016-01-01

    Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

  16. Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacylglycerol.

    PubMed Central

    Dörmann, P; Hoffmann-Benning, S; Balbo, I; Benning, C

    1995-01-01

    The galactolipids monogalactosyl and digalactosyl diacylglycerol occur in all higher plants and are the predominant lipid components of chloroplast membranes. They are thought to be of major importance to chloroplast morphology and physiology, although direct experimental evidence is still lacking. The enzymes responsible for final assembly of galactolipids are associated with the envelope membranes of plastids, and their biochemical analysis has been notoriously difficult. Therefore, we have chosen a genetic approach to study the biosynthesis and function of galactolipids in higher plants. We isolated a mutant of Arabidopsis that is deficient in digalactosyl diacylglycerol by directly screening a mutagenized M2 population for individuals with altered leaf lipid composition. This mutant carries a recessive nuclear mutation at a single locus designated dgd1. Backcrossed mutants show stunted growth, pale green leaf color, reduced photosynthetic capability, and altered thylakoid membrane ultrastructure. PMID:8535135

  17. Structure activity relationship studies on chemically non-reactive glycine sulfonamide inhibitors of diacylglycerol lipase.

    PubMed

    Chupak, Louis S; Zheng, Xiaofan; Hu, Shuanghua; Huang, Yazhong; Ding, Min; Lewis, Martin A; Westphal, Ryan S; Blat, Yuval; McClure, Andrea; Gentles, Robert G

    2016-04-01

    N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.

  18. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

    PubMed

    Astorquiza, Paula Luján; Usorach, Javier; Racagni, Graciela; Villasuso, Ana Laura

    2016-04-01

    The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

  19. Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

    PubMed

    Astorquiza, Paula Luján; Usorach, Javier; Racagni, Graciela; Villasuso, Ana Laura

    2016-04-01

    The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone. PMID:26866974

  20. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase

    PubMed Central

    Sahonero-Canavesi, Diana X.; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M.; Geiger, Otto

    2016-01-01

    Summary Phospholipids are well known for their membrane forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth. PMID:25711932

  1. Diacylglycerol-carrying lipoprotein of hemolymph of the locust and some insects.

    PubMed

    Chino, H; Kitazawa, K

    1981-09-01

    The diacylglycerol-carrying lipoprotein (DGLP) was purified from hemolymph of the locust, Locusta migratoria, by a rapid method which included a specific precipitation at low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy, and immunodiffusion. The locust DGLP molecule was almost spherical in shape with a diameter of about 130 A. The molecular weight, determined by a sedimentation equilibrium method, was approximately 580,000. The total lipid content amounted to about 40%. The lipids comprised diacylglycerol (33% of total lipid), hydrocarbon (21%), cholesterol (8%), and phospholipids (36%). The hydrocarbon fraction contained a number of n-alkanes and methylalkanes ranging from C25 to C38 in chain length. Mannose (3%) and glucosamine (0.5%) were associated with the apoprotein of DGLP. Apoprotein of locust DGLP consisted of two subunits, heavy chain (mol wt 250,000) and light chain (mol wt 85,000); carbohydrate (mannose) was associated only with the heavy chain. Tests of physiological function of DGLPs from locust, cockroach, and silkworm suggest that the insect DGLP serves multiple roles as a true carrier molecule in transporting diacylglycerol, cholesterol, and hydrocarbon from sites of storage, absorption, and synthesis to sites where these lipids are utilized as metabolic fuel, precursors for triacylglycerol and phospholipid synthesis, or structural components of cell membrane and cuticle. In addition, the insect DGLPs displayed no species-specificity in terms of the functions, whereas they were immunologically distinguishable. PMID:6795289

  2. Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases.

    PubMed

    Rodriguez, J A; Mendoza, L D; Pezzotti, F; Vanthuyne, N; Leclaire, J; Verger, R; Buono, G; Carriere, F; Fotiadu, F

    2008-04-15

    In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.

  3. Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.

    PubMed

    Sahonero-Canavesi, Diana X; Sohlenkamp, Christian; Sandoval-Calderón, Mario; Lamsa, Anne; Pogliano, Kit; López-Lara, Isabel M; Geiger, Otto

    2015-09-01

    Phospholipids are well known for their membrane-forming properties and thereby delimit any cell from the exterior world. In addition, membrane phospholipids can act as precursors for signals and other biomolecules during their turnover. Little is known about phospholipid signalling, turnover and remodelling in bacteria. Recently, we showed that a FadD-deficient mutant of Sinorhizobium meliloti, unable to convert free fatty acids to their coenzyme A derivatives, accumulates free fatty acids during the stationary phase of growth. Enzymatic activities responsible for the generation of these free fatty acids were unknown in rhizobia. Searching the genome of S. meliloti, we identified a potential lysophospholipase (SMc04041) and two predicted patatin-like phospholipases A (SMc00930, SMc01003). Although SMc00930 as well as SMc01003 contribute to the release of free fatty acids in S. meliloti, neither one can use phospholipids as substrates. Here we show that SMc01003 converts diacylglycerol to monoacylglycerol and a fatty acid, and that monoacylglycerol can be further degraded by SMc01003 to another fatty acid and glycerol. A SMc01003-deficient mutant of S. meliloti transiently accumulates diacylglycerol, suggesting that SMc01003 also acts as diacylglycerol lipase (DglA) in its native background. Expression of the DglA lipase in Escherichia coli causes lysis of cells in stationary phase of growth.

  4. Inositol Trisphosphate and Diacylglycerol Can Differentially Modulate Gene Expression in Dictyostelium

    NASA Astrophysics Data System (ADS)

    Ginsburg, Gail; Kimmel, Alan R.

    1989-12-01

    We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited. The use of selected cAMP analogs indicated that the exogenous cAMP functioned by activating the cell surface cAMP receptor and not by interacting with the regulatory subunit of the intracellular cAMP-dependent protein kinase. Although some genes in Dictyostelium appear to be regulated by intracellular cAMP, these data suggest that this is not the case for all genes regulated by cAMP. Intracellular second messengers other than cAMP may, therefore, promote the expression of these other genes. Here, we have examined inositol trisphosphate and diacylglycerol as candidates for such mediators of signal transduction. We have studied three genes that exhibit disparate modes of temporal and spatial expression during development of Dictyostelium. In shaking cultures, maximal levels of expression of each are dependent on the accumulation of or exposure to extracellular cAMP. We show that the addition of inositol trisphosphate and/or diacylglycerol to cells in shaking culture has distinct effects on the expression of each gene and, under specific conditions, can bypass the requirement for extracellular cAMP. These data suggest that extracellular cAMP interacting with its cell surface receptor may promote synthesis of inositol trisphosphate and diacylglycerol to regulate gene expression and aspects of differentiation in Dictyostelium.

  5. Molecular properties of diacylglycerol kinase-epsilon in relation to function.

    PubMed

    Jennings, William; Doshi, Sejal; D'Souza, Kenneth; Epand, Richard M

    2015-11-01

    The epsilon isoform of mammalian diacylglycerol kinase (DGKϵ) is an enzyme that associates strongly with membranes and acts on a lipid substrate, diacylglycerol. The protein has one segment that is predicted to be a transmembrane helix, but appears to interconvert between a transmembrane helix and a re-entrant helix. Despite the hydrophobicity of this segment and the fact that the lipid substrate is also hydrophobic, removal of this hydrophobic segment by truncating the protein at the amino terminus has no effect on its enzymatic activity. The amino acid sequence of the catalytic segment of DGKϵ is highly homologous to that of a bacterial DGK, DgkB. This has allowed us to predict a conformation of DGKϵ based on the known crystal structure of DgkB. An important property of DGKϵ is that it is specific for diacylglycerol species containing an arachidonoyl group. The region of DGKϵ that interacts with this group is found within the accessory domain of the protein and not in the active site nor in the hydrophobic amino terminus. The nature of the acyl chain specificity of the enzyme indicates that DGKϵ is associated with the synthesis of phosphatidylinositol. Defects or deletion of the enzyme give rise to several disease states.

  6. 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.

  7. 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

  8. The Effect of Temperature on the Level and Biosynthesis of Unsaturated Fatty Acids in Diacylglycerols of Brassica napus Leaves 1

    PubMed Central

    Williams, John P.; Khan, Mobashsher U.; Mitchell, Kirk; Johnson, Geoff

    1988-01-01

    Experiments on the effects of temperature on the levels of unsaturated fatty acids and their rates of desaturation in Brassica napus leaf lipids have shown that significant differences occur in the composition of all diacylglycerols in the leaf between plants grown at high and low temperatures. In the major thylakoid diacylglycerols, monogalactosyl-diacylglycerol and digalactosyldiacylglycerol, not only is there an increase in the level of unsaturation at low temperatures, but there is a change in the balance between molecular species of chloroplastic origin (16/18C) and cytosolic origin (18/18C). Radioactivity tracer data indicate that at low temperatures there are two distinct phases of desaturation in the fatty acids of the major diacylglycerols of these leaves. A rapid phase, which appears in plants grown at low temperatures and results in the desaturation of palmitic acid to hexadecadienoic acid and oleic acid to linoleic acid may explain the high levels of unsaturated fatty acids found in the leaf diacylglycerols from plants grown at low temperatures. The appearance of this rapid phase is controlled by the temperature at which the plant is grown and is not subject to rapid variations in environmental temperature. PMID:16666243

  9. Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes

    PubMed Central

    Covés, Jacques; Joyard, Jacques; Douce, Roland

    1988-01-01

    We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-β-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose. PMID:16593955

  10. Interfacial partitioning of a loop hinge residue contributes to diacylglycerol affinity of conserved region 1 domains.

    PubMed

    Stewart, Mikaela D; Cole, Taylor R; Igumenova, Tatyana I

    2014-10-01

    Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826-830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities.

  11. 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.

  12. Biosynthesis of Phytosterol Esters: Identification of a Sterol O-Acyltransferase in Arabidopsis1[OA

    PubMed Central

    Chen, Qilin; Steinhauer, Lee; Hammerlindl, Joe; Keller, Wilf; Zou, Jitao

    2007-01-01

    Fatty acyl esters of phytosterols are a major form of sterol conjugates distributed in many parts of plants. In this study we report an Arabidopsis (Arabidopsis thaliana) gene, AtSAT1 (At3g51970), which encodes for a novel sterol O-acyltransferase. When expressed in yeast (Saccharomyces cerevisiae), AtSAT1 mediated production of sterol esters enriched with lanosterol. Enzyme property assessment using cell-free lysate of yeast expressing AtSAT1 suggested the enzyme preferred cycloartenol as acyl acceptor and saturated fatty acyl-Coenyzme A as acyl donor. Taking a transgenic approach, we showed that Arabidopsis seeds overexpressing AtSAT1 accumulated fatty acyl esters of cycloartenol, accompanied by substantial decreases in ester content of campesterol and β-sitosterol. Furthermore, fatty acid components of sterol esters from the transgenic lines were enriched with saturated and long-chain fatty acids. The enhanced AtSAT1 expression resulted in decreased level of free sterols, but the total sterol content in the transgenic seeds increased by up to 60% compared to that in wild type. We conclude that AtSAT1 mediates phytosterol ester biosynthesis, alternative to the route previously described for phospholipid:sterol acyltransferase, and provides the molecular basis for modification of phytosterol ester level in seeds. PMID:17885082

  13. Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

    PubMed Central

    Pérez-Gil, J; Martín, J; Acebal, C; Arche, R

    1990-01-01

    Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. PMID:2241908

  14. Identification of a palmitoyl acyltransferase required for protein sorting to the flagellar membrane.

    PubMed

    Emmer, Brian T; Souther, Christina; Toriello, Krista M; Olson, Cheryl L; Epting, Conrad L; Engman, David M

    2009-03-15

    Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms. PMID:19240115

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

    PubMed

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

    2010-02-01

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

  16. 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

  17. Biochemical and Structural Study of the Atypical Acyltransferase Domain from the Mycobacterial Polyketide Synthase Pks13*

    PubMed Central

    Bergeret, Fabien; Gavalda, Sabine; Chalut, Christian; Malaga, Wladimir; Quémard, Annaïk; Pedelacq, Jean-Denis; Daffé, Mamadou; Guilhot, Christophe; Mourey, Lionel; Bon, Cécile

    2012-01-01

    Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction. PMID:22825853

  18. Structural characterization of phosphatidylcholine-diacylglycerol system by neutron scattering and X-ray diffraction

    NASA Astrophysics Data System (ADS)

    Takahashi, H.; Nagura, K.; Imai, M.; Matsushita, Y.; Hatta, I.

    Diacylglycerol (DAG) is recognized as one of the most important lipids for biological functions of cell membranes. In order to understand the functions of DAG, it is indispensable to study the effect of DAG on phosphatidylcholine (PC), which is a main lipid component of biomembranes. Here we report neutron-scattering data of sonicated PC/DAG vesicles and X-ray-diffraction data of oriented PC/DAG multilamellar systems. These data imply that addition of DAG induces a change in the tilt angle of lipid molecules and that, as a result, an increase of the membrane thickness is induced.

  19. Transcription factor Reb1p regulates DGK1-encoded diacylglycerol kinase and lipid metabolism in Saccharomyces cerevisiae.

    PubMed

    Qiu, Yixuan; Fakas, Stylianos; Han, Gil-Soo; Barbosa, Antonio Daniel; Siniossoglou, Symeon; Carman, George M

    2013-10-01

    In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

  20. 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

  1. Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

    PubMed

    Mandon, E C; Ehses, I; Rother, J; van Echten, G; Sandhoff, K

    1992-06-01

    Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%. PMID:1317856

  2. 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.

  3. 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

  4. 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

  5. The expression of diacylglycerol kinase theta during the organogenesis of mouse embryos

    PubMed Central

    2013-01-01

    Background Diacylglycerol kinase (DGK) is a key enzyme that regulates diacylglycerol (DG) turnover and is involved in a variety of physiological functions. The isoform DGKθ has a unique domain structure and is the sole member of type V DGK. To reveal the spatial and temporal expression of DGKθ we performed immunohistochemical staining on paraffin sections of mouse embryos. Results At an early stage of development (E10.5 and 11.5), the expression of DGKθ was prominently detected in the brain, spinal cord, dorsal root ganglion, and limb bud, and was also moderately detected in the bulbus cordis and the primordium of the liver and gut. At later stages (E12.5 and 14.5), DGKθ expression persisted or increased in the neocortex, epithalamus, hypothalamus, medulla oblongata, and pons. DGKθ was also evident in the epidermis, and nearly all epithelia of the oropharyngeal membrane, digestive tract, and bronchea. At prenatal developmental stages (E16.5 and E18.5), the expression pattern of DGKθ was maintained in the central nervous system, intestine, and kidney, but was attenuated in the differentiated epidermis. Conclusion These results suggest that DGKθ may play important physiological roles not only in the brain, but also in diverse organs and tissues during the embryonic stages. PMID:24079595

  6. Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

    PubMed

    Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-10-01

    Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth. PMID:27423518

  7. Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland.

    PubMed

    Hozumi, Yasukazu; Akimoto, Ryo; Suzuki, Akihito; Otani, Koichi; Watanabe, Masahiko; Goto, Kaoru

    2015-11-01

    Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGKγ localizes to the Golgi complex, DGKε to the plasma membrane, and DGKζ to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.

  8. Diacylglycerol kinase α regulates tubular recycling endosome biogenesis and major histocompatibility complex class I recycling.

    PubMed

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2014-11-14

    Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.

  9. Comprehensive genomic analysis and expression profiling of diacylglycerol kinase gene family in Malus prunifolia (Willd.) Borkh.

    PubMed

    Li, Yali; Tan, Yanxiao; Shao, Yun; Li, Mingjun; Ma, Fengwang

    2015-05-01

    Diacylglycerol kinase (DGK) is a pivotal enzyme that phosphorylates diacylglycerol (DAG) to form phosphatidic acid (PA). The production of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a critical signaling process in animal and plant cells. Next to PLD, DGK is the second most important generator of PA in biotic and abiotic stress responses. We identified 8 DGK members within the apple genome and all of their putative proteins contain one DGK catalytic domain and one DGK accessory domain. Four coding sequences were confirmed by cloning from Malus prunifolia. Phylogenetic and gene structure analyses showed that the apple DGK genes could be assigned to Clusters I, II, or III. Expression analysis of 6 of them revealed that their transcript levels were highest in stems. Some apple DGK genes were also significantly up-regulated in response to salt and drought stresses. This suggested their possible roles in plant defenses against environmental challenges. As a first step toward genome-wide analyses of the DGK genes in woody plants, our results imply that apple DGK genes are involved in the signaling of stress responses. These findings will contribute to further functional dissection of this gene family.

  10. Diacylglycerol Kinase ζ Is a Target To Enhance NK Cell Function.

    PubMed

    Yang, Enjun; Singh, Brenal K; Paustian, Amanda M Schmidt; Kambayashi, Taku

    2016-08-01

    Enhancement of NK cell function could be beneficial in treatment of a variety of tumors and infections. However, efforts to improve NK cell function by disrupting negative regulators that target proximal signaling pathways paradoxically results in hyporesponsive rather than hyperresponsive NK cells. In this study, we demonstrate that genetic deletion of diacylglycerol kinase (DGK)ζ, a negative regulator of diacylglycerol-mediated signaling, has the desired effect of enhancing NK cell function due to its distal position in the activating receptor-mediated signaling cascade. Upon stimulation through multiple activating receptors, NK cells from mice lacking DGKζ display increased cytokine production and degranulation in an ERK-dependent manner. Additionally, they have improved cytotoxic functions against tumor cell lines. The enhancement of NK cell function by DGKζ deficiency is NK cell-intrinsic and developmentally independent. Importantly, DGKζ deficiency does not affect inhibitory NK cell receptor expression or function. Thus, DGKζ knockout mice display improved missing self recognition, as evidenced by enhanced rejection of a TAP-deficient tumor in vivo. We propose that enzymes that negatively regulate distal activating receptor signaling pathways such as DGKζ represent novel targets for augmenting the therapeutic potential of NK cells.

  11. Behavioral and pharmacological phenotypes of brain-specific diacylglycerol kinase δ-knockout mice.

    PubMed

    Usuki, Takako; Takato, Tamae; Lu, Qiang; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-10-01

    Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth.

  12. Distinct properties of the two isoforms of CDP-diacylglycerol synthase.

    PubMed

    D'Souza, Kenneth; Kim, Yeun Ju; Balla, Tamas; Epand, Richard M

    2014-12-01

    CDP-diacylglycerol synthases (CDS) are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain-dependent, with the strongest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain-dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the strongest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains.

  13. [Synthesis of diacylglycerol using immoblized regiospecific lipase in continuously operated fixed bed reactors].

    PubMed

    Meng, Xiang-He; Sun, Pei-Long; Yang, Kai; He, Rong-Jun; Mao, Zhong-Gui

    2005-05-01

    Diacylglycerol, DAG, because of its multifunctional and nutritional properties, attracted considerable attention recently. Enzymatic synthesis of diacylglycerols from linoleic acid was investigated in a solvent-free reaction in a continuously operated fixed bed reactors containing Lipozyme RM IM. By appropriate manipulation of the fluid-residence time, the relative proportions of the various acylglycerols in the effluent stream can be controlled. In addition, the presence of excess glycerol is effective for the removal of water produced during the esterification reactions. Under the conditions of molar ratio of linoleic acid to glycerol of 0.5, the immoblized enzyme maintained high stability and allowed the reaction to continue for 10 days without significant deterioration in enzyme activity. It was determined that the conversion of fatty acid, content of 1,3-DAG and volume efficiency of reactor reached optima under the conditions: a packaged-bed reactor(with a ratio of packed length to inner diameter of 7.8), reacting temperature at 65 degrees C, molar ratio of linoleic acid to glycerol of 0.5, and feeding flow rate of 1.2 mL/min.

  14. Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids.

    PubMed

    Jiménez-Dalmaroni, Maximiliano J; Radcliffe, Catherine M; Harvey, David J; Wormald, Mark R; Verdino, Petra; Ainge, Gary D; Larsen, David S; Painter, Gavin F; Ulevitch, Richard; Beutler, Bruce; Rudd, Pauline M; Dwek, Raymond A; Wilson, Ian A

    2015-02-01

    TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED. PMID:24591200

  15. Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function

    PubMed Central

    Krishna, Sruti; Zhong, Xiao-Ping

    2013-01-01

    Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αβ T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors. PMID:23847619

  16. Mass spectrometry of the lithium adducts of diacylglycerols containing hydroxy FA in castor oil and two normal FA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass ...

  17. Diacylglycerol lipase regulates lifespan and oxidative stress response by inversely modulating TOR signaling in Drosophila and C. elegans.

    PubMed

    Lin, Yen-Hung; Chen, Yi-Chun; Kao, Tzu-Yu; Lin, Yi-Chun; Hsu, Tzu-En; Wu, Yi-Chun; Ja, William W; Brummel, Theodore J; Kapahi, Pankaj; Yuh, Chiou-Hwa; Yu, Lin-Kwei; Lin, Zhi-Han; You, Ru-Jing; Jhong, Yi-Ting; Wang, Horng-Dar

    2014-08-01

    Target of rapamycin (TOR) signaling is a nutrient-sensing pathway controlling metabolism and lifespan. Although TOR signaling can be activated by a metabolite of diacylglycerol (DAG), phosphatidic acid (PA), the precise genetic mechanism through which DAG metabolism influences lifespan remains unknown. DAG is metabolized to either PA via the action of DAG kinase or 2-arachidonoyl-sn-glycerol by diacylglycerol lipase (DAGL). Here, we report that in Drosophila and Caenorhabditis elegans, overexpression of diacylglycerol lipase (DAGL/inaE/dagl-1) or knockdown of diacylglycerol kinase (DGK/rdgA/dgk-5) extends lifespan and enhances response to oxidative stress. Phosphorylated S6 kinase (p-S6K) levels are reduced following these manipulations, implying the involvement of TOR signaling. Conversely, DAGL/inaE/dagl-1 mutants exhibit shortened lifespan, reduced tolerance to oxidative stress, and elevated levels of p-S6K. Additional results from genetic interaction studies are consistent with the hypothesis that DAG metabolism interacts with TOR and S6K signaling to affect longevity and oxidative stress resistance. These findings highlight conserved metabolic and genetic pathways that regulate aging.

  18. Diacylglycerol kinase zeta deficiency in a non-CD4+T cell compartment leads to increased peanut hypersensitivity

    PubMed Central

    Kulis, Mike; Wan, Chi-Keung; Gorentla, Balachandra K.; Burks, A. Wesley; Zhong, Xiao-Ping

    2011-01-01

    Summary Peanut sensitization in diacylglycerol kinase zeta (DGKζ) deficient mice led to elevated peanut-IgE levels and severe anaphylaxis. DGKζ deficient CD4+T cells did not account for the phenotype. Future studies will determine which immune lineage caused increased food hypersensitivity. PMID:21439625

  19. Quantification of the molecular species of diacylglycerols,triacylglycerols and tetraacylglycerols in lesquerella (Physaria fendleri) oil by HPLC and MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ten diacylglycerols (DAG), 74 triacylglycerols (TAG) and 13 tetraacylglycerols in the seed oil of Physaria fendleri were recently identified by HPLC and MS. These acylglycerols (AG) were quantified by HPLC with evaporative light scattering detector and electrospray ionization mass spectrometry of th...

  20. Lysophosphatidylcholine metabolism to 1,2-diacylglycerol in lymphoblasts: Involvement of a phosphatidylcholine-hydrolyzing phospholipase C

    SciTech Connect

    Nishijima, J.; Wright, T.M.; Hoffman, R.D.; Liao, F.; Symer, D.E.; Shin, H.S. )

    1989-04-04

    The authors have previously described the chemoattraction of lymphoblasts by lysophosphatidylcholine. In studying the mechanism of chemoattraction it was found that lysophosphatidylcholine was metabolized to 1,2-diacylglycerol by the lymphoblastic cell line 6C3HED. One route of metabolism involves the acylation of lysophosphatidylcholine to phosphatidylcholine with subsequent hydrolysis to 1,2-diacylglycerol and phosphocholine by the action of phospholipase C. The increase in cellular 1,2-diacylglycerol was established by metabolic experiments using ({sup 14}C)glycerol-labeled lysophosphatidylcholine and by mass measurements of 1,2-diacylglycerol. The presence of a phosphatidylcholine-hydrolyzing phospholipase C was confirmed in 6C3HED cell homogenates. In intact cells, lysophosphatidylcholine induced a pattern of protein phosphorylation similar to those of 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate, two known activators of protein kinase C. This pathway of lysophosphatidylcholine metabolism, which involves a phosphatidylcholine-hydrolyzing phospholipase C, may be important in the activation of protein kinase C independent of inositol phospholipid hydrolysis.

  1. Assignment of the human diacylglycerol kinase 4 (DAGK4) gene to chromosome 4p16.3

    SciTech Connect

    Endele, S.; Zabel, B.; Winterpacht, A.

    1996-04-01

    This report describes the localization of the human gene for diacylglycerol kinase 4 (DAGK4) to human chromosome 4p16.3 using an exon amplification scheme. It also discusses the possible implications of the chromosomal location of this gene in certain hereditary malignancies. 9 refs., 1 fig.

  2. Novel Acylphosphate Mimics that Target PlsY, an Essential Acyltransferase in Gram-Positive Bacteria

    PubMed Central

    Grimes, Kimberly D.; Lu, Ying-Jie; Zhang, Yong-Mei; Luna, Vicki A.; Hurdle, Julian G.; Carson, Elizabeth I.; Qi, Jianjun; Kudrimoti, Sucheta; Rock, Charles O.

    2009-01-01

    PlsY is a recently discovered acyltransferase that executes an essential step in membrane phospholipid biosynthesis in Gram-positive bacteria. Using a bioisosteric replacement approach to generate substrate-based inhibitors of PlsY as potential novel antibacterial agents, a series of stabilized acylphosphate mimetics, including acylphosphonates, acyl αα,-difluoromethyl phosphonates, acyl phosphoramides, reverse amide phosphonates, acylsulfamates and acylsulfamides were designed and synthesized. Several acyl phosphonates, phosphoramides and sulfamates were identified as inhibitors of PlsY from Streptococcus pneumoniae and Bacillus anthracis. As anticipated, these inhibitors were competitive inhibitors with respect to the acylphosphate substrate. Antimicrobial testing showed the inhibitors to have generally weak anti Gram-positive activity with the exception of some acyl phosphonates, reverse amide phosphonates, and acylsulfamates that had potent activity against multiple strains of Bacillus anthracis. PMID:19016283

  3. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal.

    PubMed

    Chamoun, Z; Mann, R K; Nellen, D; von Kessler, D P; Bellotto, M; Beachy, P A; Basler, K

    2001-09-14

    One of the most dominant influences in the patterning of multicellular embryos is exerted by the Hedgehog (Hh) family of secreted signaling proteins. Here, we identify a segment polarity gene in Drosophila melanogaster, skinny hedgehog (ski), and show that its product is required in Hh-expressing cells for production of appropriate signaling activity in embryos and in the imaginal precursors of adult tissues. The ski gene encodes an apparent acyltransferase, and we provide genetic and biochemical evidence that Hh proteins from ski mutant cells retain carboxyl-terminal cholesterol modification but lack amino-terminal palmitate modification. Our results suggest that ski encodes an enzyme that acts within the secretory pathway to catalyze amino-terminal palmitoylation of Hh, and further demonstrate that this lipid modification is required for the embryonic and larval patterning activities of the Hh signal.

  4. 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.

  5. 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.

  6. 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

  7. Measurement of lecithin-cholesterol acyltransferase activity with the use of a Peptide-proteoliposome substrate.

    PubMed

    Vaisman, Boris L; Remaley, Alan T

    2013-01-01

    Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme responsible for the esterification of free cholesterol on plasma lipoproteins, which is a key step in the reverse cholesterol transport pathway. The measurement of plasma LCAT activity not only is important in the diagnosis of patients with genetic or acquired LCAT deficiency but is also valuable in calculating cardiovascular risk, as well as in research studies of lipoprotein metabolism. In this chapter, we describe a convenient LCAT assay based on the use of an apoA-I mimetic peptide. The proteoliposome substrate used in this assay for LCAT is easily made with the peptide and can be stored by deep freezing without significant loss of activity. PMID:23912995

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

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

    PubMed

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

    2007-10-01

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

  14. 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.

  15. 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

  16. 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

  17. Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase*

    PubMed Central

    Lang, Christina; Rastew, Elena; Hermes, Björn; Siegbrecht, Enrico; Ahrends, Robert; Banerji, Sangeeta; Flieger, Antje

    2012-01-01

    Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated. PMID:22582391

  18. 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.

  19. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels. PMID:25112873

  20. Diacylglycerol kinase δ phosphorylates phosphatidylcholine-specific phospholipase C-dependent, palmitic acid-containing diacylglycerol species in response to high glucose levels.

    PubMed

    Sakai, Hiromichi; Kado, Sayaka; Taketomi, Akinobu; Sakane, Fumio

    2014-09-19

    Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.

  1. Hormone-sensitive hepatic Na/sup +/-pump: evidence for regulation by diacylglycerol and tumor promoters

    SciTech Connect

    Lynch, C.J.; Wilson, P.B.; Blackmore, P.F.; Exton, J.H.

    1986-11-05

    Ouabain-sensitive /sup 86/Rb/sup +/ uptake by isolated rat hepatocytes was studied to elucidate how Ca/sup 2 +/-mobilizing hormones stimulate the Na/sup +/-pump. Stimulation of this uptake was observed with concentrations of vasopressin ((8-arginine)vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca/sup 2 +/ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca/sup 2 +/, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca/sup 2 +/-free Krebs-Henseleit buffer, Na/sup +/-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca/sup 2 +/. Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca/sup 2 +/, but only modestly attenuated AVP-stimulated Na/sup +/-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na/sup +//K/sup +/-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na/sup +/-pump in the absence of changes in cytosolic or total cellular Ca/sup 2 +/ levels. Stimulation of the Na/sup +/-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na/sup +/-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na/sup +//H/sup +/ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na/sup +//K/sup +/-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca/sup 2 +/-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.

  2. Synthesis and Biological Evaluation of Several Bryostatin Analogues Bearing a Diacylglycerol Lactone C-Ring.

    PubMed

    Baumann, David O; McGowan, Kevin M; Kedei, Noemi; Peach, Megan L; Blumberg, Peter M; Keck, Gary E

    2016-09-01

    As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings. PMID:27494208

  3. Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cardiomyocyte hypertrophy.

    PubMed

    Takeishi, Yasuchika; Goto, Kaoru; Kubota, Isao

    2007-09-01

    Diacylglycerol (DAG) kinase (DGK) phosphorylates and converts DAG to phosphatidic acid. DGK regulates cellular DAG levels and attenuates DAG signaling. The 10 mammalian DGK isoforms have been identified to date. In cardiac myocytes, DGKalpha, epsilon, and zeta are expressed, and DGKzeta is the predominant isoform. DGKzeta inhibits protein kinase C (PKC) activation and subsequent hypertrophic programs in response to endothelin-1 (ET-1) in neonatal rat cardiomyocytes. DGKzeta blocks cardiac hypertrophy induced by G protein-coupled receptor agonists and pressure overload in vivo. DGKzeta attenuates ventricular remodeling and improves survival after myocardial infarction. These data provide a novel insight for subcellular mechanisms of cardiac hypertrophy and heart failure, and DGKzeta may be a new therapeutic target to prevent cardiac hypertrophy and progression to heart failure. PMID:17659347

  4. CDP-diacylglycerol synthetase-controlled phosphoinositide availability limits VEGFA signaling and vascular morphogenesis

    PubMed Central

    Pan, Weijun; Pham, Van N.; Stratman, Amber N.; Castranova, Daniel; Kamei, Makoto; Kidd, Kameha R.; Lo, Brigid D.; Shaw, Kenna M.; Torres-Vazquez, Jesus; Mikelis, Constantinos M.; Gutkind, J. Silvio; Davis, George E.

    2012-01-01

    Understanding the mechanisms that regulate angiogenesis and translating these into effective therapies are of enormous scientific and clinical interests. In this report, we demonstrate the central role of CDP-diacylglycerol synthetase (CDS) in the regulation of VEGFA signaling and angiogenesis. CDS activity maintains phosphoinositide 4,5 bisphosphate (PIP2) availability through resynthesis of phosphoinositides, whereas VEGFA, mainly through phospholipase Cγ1, consumes PIP2 for signal transduction. Loss of CDS2, 1 of 2 vertebrate CDS enzymes, results in vascular-specific defects in zebrafish in vivo and failure of VEGFA-induced angiogenesis in endothelial cells in vitro. Absence of CDS2 also results in reduced arterial differentiation and reduced angiogenic signaling. CDS2 deficit-caused phenotypes can be successfully rescued by artificial elevation of PIP2 levels, and excess PIP2 or increased CDS2 activity can promote excess angiogenesis. These results suggest that availability of CDS-controlled resynthesis of phosphoinositides is essential for angiogenesis. PMID:22649102

  5. Protein kinase C modulates the motional properties of its lipid cofactor DPH-diacylglycerol

    NASA Astrophysics Data System (ADS)

    Pap, Eward; Borst, Jan-Willem; Ketelaars, Martjin; van Hoek, Arie; Visser, Antonie J. W. G.

    1994-08-01

    The motional properties of DPH labelled diacylglycerol (DG) in vesicles have been investigated in the absence and presence of its biological target: protein kinase C (PKC). In the absence of PKC the extent of ordering and rotational dynamics of DPH-DG turned out to be considerably different from those of DPH labelled phosphatidylcholine (DPH-PC). When DPH-DG was dispersed in membranes containing 10 mole % of phosphatidylserine (PS), addition of PKC led to an immobilization as judged from a slower fluorescence anisotropy decay. This effect was not seen when PS was replaced by PC or in the absence of calcium indicating that negatively charged lipids and calcium are required for interaction between PKC and DPH-DG. Furthermore, the specificity of the interaction of PKC with DPH-DG was compared with that of the control choline lipid DPH-PC.

  6. Optimization of mono and diacylglycerols production from enzymatic glycerolysis in solvent-free systems.

    PubMed

    Valério, Alexsandra; Rovani, Suzimara; Treichel, Helen; de Oliveira, Débora; Oliveira, J Vladimir

    2010-09-01

    This work reports solvent-free enzymatic glycerolysis of olive oil with an immobilized lipase (Novozym 435) using Tween 40, Tween 65, Tween 80, Tween 85, Triton X-100, and soy lecithin as surfactants. The first step was the screening of two potential surfactants for Monoacylglycerol (MAG) and Diacylglycerol (DAG) production with a pre-established operating condition and 2 h of reaction time. Afterwards, a sequential experimental design strategy was carried out in order to optimize MAG and DAG production using Tween 65 and Triton X-100 as surfactants. The operating conditions that optimized MAG and DAG yields were 70 degrees C, stirring rate of 600 rpm, glycerol:olive oil molar ratio of 6:1, 16 wt% of surfactant Tween 65 and 9.0 wt% of Novozym 435, leading to a content of 26 and 17 wt% of MAG and DAG, respectively.

  7. Preparation of mono- and diacylglycerols by enzymatic esterification of glycerol with conjugated linoleic acid in hexane.

    PubMed

    Martinez, C E; Vinay, J C; Brieva, R; Hill, C G; Garcia, H S

    2005-04-01

    Esterification of glycerol with conjugated linoleic acid (CLA) was carried out in hexane. Lipase from Rhizomucor miehei provided a high degree of esterification (80%) in 8 h at 50 degrees C when used at 15% (w/w) in a system containing a 1:2 molar ratio of glycerol to free fatty acids. Esterification levels >80% were obtained in 8 h at 40 degrees C with 15% (w/w) lipase from Candida antarctica at the same molar ratio of reactants. The extent of esterification of CLA was >90% after 4 h of reaction at 50 degrees C with a 5% (w/w) loading of either R. miehei or C. antarctica lipase, together with a 1:1 molar ratio of substrates. Both enzymes incorporated the original CLA as acylglycerol residues in primarily 1,3-diacylglycerol and 1-monoacylglycerol. The CLA-rich acylglycerols can be employed as emulsifiers or as substitutes for natural fats and oils.

  8. Soluble human TLR2 ectodomain binds diacylglycerol from microbial lipopeptides and glycolipids

    PubMed Central

    Jiménez-Dalmaroni, Maximiliano J; Radcliffe, Catherine M; Harvey, David J; Wormald, Mark R; Verdino, Petra; Ainge, Gary D; Larsen, David S; Painter, Gavin F; Ulevitch, Richard; Beutler, Bruce; Rudd, Pauline M; Dwek, Raymond A; Wilson, Ian A

    2015-01-01

    Toll-like receptors (TLRs) are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system, and its biochemical as well as ligand binding properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid (LTA) from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chain containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED. The design of novel inhibitors of TLR2, based on their ability to bind to TLR2 but not activate the TLR2 signaling pathway, may lead to the development of novel treatments for septic shock caused by Gram- positive bacteria. PMID:24591200

  9. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  10. A novel live cell assay to measure diacylglycerol lipase α activity

    PubMed Central

    Singh, Praveen K.; Markwick, Rachel; Howell, Fiona V.; Williams, Gareth; Doherty, Patrick

    2016-01-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays. PMID:27013337

  11. Fragile X Mental Retardation Protein (FMRP) controls diacylglycerol kinase activity in neurons.

    PubMed

    Tabet, Ricardos; Moutin, Enora; Becker, Jérôme A J; Heintz, Dimitri; Fouillen, Laetitia; Flatter, Eric; Krężel, Wojciech; Alunni, Violaine; Koebel, Pascale; Dembélé, Doulaye; Tassone, Flora; Bardoni, Barbara; Mandel, Jean-Louis; Vitale, Nicolas; Muller, Dominique; Le Merrer, Julie; Moine, Hervé

    2016-06-28

    Fragile X syndrome (FXS) is caused by the absence of the Fragile X Mental Retardation Protein (FMRP) in neurons. In the mouse, the lack of FMRP is associated with an excessive translation of hundreds of neuronal proteins, notably including postsynaptic proteins. This local protein synthesis deregulation is proposed to underlie the observed defects of glutamatergic synapse maturation and function and to affect preferentially the hundreds of mRNA species that were reported to bind to FMRP. How FMRP impacts synaptic protein translation and which mRNAs are most important for the pathology remain unclear. Here we show by cross-linking immunoprecipitation in cortical neurons that FMRP is mostly associated with one unique mRNA: diacylglycerol kinase kappa (Dgkκ), a master regulator that controls the switch between diacylglycerol and phosphatidic acid signaling pathways. The absence of FMRP in neurons abolishes group 1 metabotropic glutamate receptor-dependent DGK activity combined with a loss of Dgkκ expression. The reduction of Dgkκ in neurons is sufficient to cause dendritic spine abnormalities, synaptic plasticity alterations, and behavior disorders similar to those observed in the FXS mouse model. Overexpression of Dgkκ in neurons is able to rescue the dendritic spine defects of the Fragile X Mental Retardation 1 gene KO neurons. Together, these data suggest that Dgkκ deregulation contributes to FXS pathology and support a model where FMRP, by controlling the translation of Dgkκ, indirectly controls synaptic proteins translation and membrane properties by impacting lipid signaling in dendritic spine.

  12. Plasma lipidomic profile signature of hypertension in Mexican American families: specific role of diacylglycerols.

    PubMed

    Kulkarni, Hemant; Meikle, Peter J; Mamtani, Manju; Weir, Jacquelyn M; Barlow, Christopher K; Jowett, Jeremy B; Bellis, Claire; Dyer, Thomas D; Johnson, Matthew P; Rainwater, David L; Almasy, Laura; Mahaney, Michael C; Comuzzie, Anthony G; Blangero, John; Curran, Joanne E

    2013-09-01

    Both as a component of metabolic syndrome and as an independent entity, hypertension poses a continued challenge with regard to its diagnosis, pathogenesis, and treatment. Previous studies have documented connections between hypertension and indicators of lipid metabolism. Novel technologies, such as plasma lipidomic profiling, promise a better understanding of disorders in which there is a derangement of the lipid metabolism. However, association of plasma lipidomic profiles with hypertension in a high-risk population, such as Mexican Americans, has not been evaluated before. Using the rich data and sample resource from the ongoing San Antonio Family Heart Study, we conducted plasma lipidomic profiling by combining high-performance liquid chromatography with tandem mass spectroscopy to characterize 319 lipid species in 1192 individuals from 42 large and extended Mexican American families. Robust statistical analyses using polygenic regression models, liability threshold models, and bivariate trait analyses implemented in the SOLAR software were conducted after accounting for obesity, insulin resistance, and relative abundance of various lipoprotein fractions. Diacylglycerols, in general, and the DG 16:0/22:5 and DG 16:0/22:6 lipid species, in particular, were significantly associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), as well as liability of incident hypertension measured during 7140.17 person-years of follow-up. Four lipid species, including the DG 16:0/22:5 and DG 16:0/22:6 species, showed significant genetic correlations with the liability of hypertension in bivariate trait analyses. Our results demonstrate the value of plasma lipidomic profiling in the context of hypertension and identify disturbance of diacylglycerol metabolism as an independent biomarker of hypertension. PMID:23798346

  13. A key role for diacylglycerol lipase-alpha in metabotropic glutamate receptor-dependent endocannabinoid mobilization.

    PubMed

    Jung, Kwang-Mook; Astarita, Giuseppe; Zhu, Chenggang; Wallace, Matthew; Mackie, Ken; Piomelli, Daniele

    2007-09-01

    Activation of group I metabotropic glutamate (mGlu) receptors recruits the endocannabinoid system to produce both short- and long-term changes in synaptic strength in many regions of the brain. Although there is evidence that the endocannabinoid 2-arachidonoylglycerol (2-AG) mediates this process, the molecular mechanism underlying 2-AG mobilization remains unclear. In the present study, we used a combination of genetic and targeted lipidomic approaches to investigate the role of the postsynaptic membrane-associated lipase, diacylglycerol lipase type-alpha (DGL-alpha), in mGlu receptor-dependent 2-AG mobilization. DGL-alpha overexpression in mouse neuroblastoma Neuro-2a cells increased baseline 2-AG levels. This effect was accompanied by enhanced utilization of the 2-AG precursor 1-stearoyl,2-arachidonoyl-sn-glycerol and increased accumulation of the 2-AG breakdown product arachidonic acid. A similar, albeit less marked response was observed with other unsaturated and polyunsaturated monoacylglycerols, 1,2-diacylglycerols, and fatty acids. Silencing of DGL-alpha by RNA interference elicited lipidomic changes opposite those of DGL-alpha overexpression and abolished group I mGlu receptor-dependent 2-AG mobilization. Coimmunoprecipitation and site-directed mutagenesis experiments revealed that DGL-alpha interacts, via a PPxxF domain, with the coiled-coil (CC)-Homer proteins Homer-1b and Homer-2, two components of the molecular scaffold that enables group I mGlu signaling. DGL-alpha mutants that do not bind Homer maintained their ability to generate 2-AG in intact cells but failed to associate with the plasma membrane. The findings indicate that DGL-alpha mediates group I mGlu receptor-induced 2-AG mobilization. They further suggest that the interaction of CC-Homer with DGL-alpha is necessary for appropriate function of this lipase.

  14. Pharmacological evidence for the involvement of diacylglycerol lipase in depolarization-induced endocanabinoid release.

    PubMed

    Hashimotodani, Yuki; Ohno-Shosaku, Takako; Maejima, Takashi; Fukami, Kiyoko; Kano, Masanobu

    2008-01-01

    Depolarization-induced suppression of inhibition (DSI) or excitation (DSE) is a well-known form of endocannabinoid-mediated short-term plasticity that is induced by postsynaptic depolarization. It is generally accepted that DSI/DSE is triggered by Ca(2+) influx through voltage-gated Ca(2+) channels. It is also demonstrated that DSI/DSE is mediated by 2-arachidonoylglycerol (2-AG). However, how Ca(2+) induces 2-AG production is still unclear. In the present study, we investigated molecular mechanisms underlying the Ca(2+)-driven 2-AG production. Using cannabinoid-sensitive inhibitory synapses of cultured hippocampal neurons, we tested several inhibitors for enzymes that are supposed to be involved in 2-AG metabolism. The chemicals we tested include inhibitors for phospholipase C (U73122 and ET-18), diacylglycerol kinase (DGK inhibitor 1), phosphatidic acid phosphohydrolase (propranolol), and diacylglycerol lipase (DGL; RHC-80267 and tetrahydrolipstatin (THL)). However, unfavorable side effects were observed with these inhibitors, except for THL. Furthermore, we found that RHC-80267 hardly inhibited the endocannabinoid release driven by G(q/11)-coupled receptors, which is thought to be DGL-dependent. By contrast, THL exhibited no side effects as long as we tested, and was confirmed to inhibit the DGL-dependent process. Using THL as a DGL inhibitor, we demonstrated that DGL is involved in both hippocampal DSI and cerebellar DSE. To test a possible involvement of PLCdelta in DSI, we examined hippocampal DSI in PLCdelta1, delta3 and delta4-knockout mice. However, there was no significant difference in the DSI magnitude between these knockout mice and wild-type mice. The present study clearly shows that DGL is a prerequisite for DSI/DSE. The enzymes yielding DG remain to be determined.

  15. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

  16. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  17. A novel live cell assay to measure diacylglycerol lipase α activity.

    PubMed

    Singh, Praveen K; Markwick, Rachel; Howell, Fiona V; Williams, Gareth; Doherty, Patrick

    2016-06-01

    Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

  18. Reciprocal regulation of p53 and NF-κB by diacylglycerol kinase ζ.

    PubMed

    Tanaka, Toshiaki; Tsuchiya, Rieko; Hozumi, Yasukazu; Nakano, Tomoyuki; Okada, Masashi; Goto, Kaoru

    2016-01-01

    Diacylglycerol kinase (DGK) participates in lipid mediated-signal transduction. It phosphorylates diacylglycerol (DG) to phosphatidic acid (PA), thereby regulating the balanced control of these second messenger actions. Previous reports have described that one DGK family, DGKζ, is closely involved in stress responses under various conditions. Cellular stress response, a physiological process enabling cells to cope with an altered environment, is finely tuned through various signaling cascades and their molecular crosstalk. The major components of stress response are p53 and NF-κB. p53 generally serves as a proapoptotic transcriptional factor, whereas NF-κB promotes resistance to programmed cell death under most circumstances. Recent studies have suggested that DGKζ facilitates p53 degradation in cytoplasm through ubiquitin proteasome system and that DGKζ deletion upregulates p53 protein levels under basal and DNA-damage conditions. Counter-intuitively, however, DGKζ deletion suppresses p53 transcriptional activity despite increased p53 levels. In contrast, DGKζ knockdown engenders enhancement of NF-κB pathway in response to cytokines such as TNF-α and IL-1β. In response to these cytokines, DGKζ downregulation accelerates phosphorylation of the p65 subunit and its nuclear translocation, thereby enhancing NF-κB transcriptional activity. Furthermore, DGKζ deficiency is shown to promote increased association of p65 subunit with the transcriptional cofactor CBP. It is particularly interesting that this association is observed even under basal conditions in the absence of stimulation. These findings suggest that DGKζ plays a role in sequestration of the limiting pool of CBP/p300 between the NF-κB p65 subunit and p53, and that DGKζ downregulation shifts CBP/p300 toward the NF-κB subunit to regulate reciprocally antagonistic phenotypes of these transcription factors.

  19. Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish triggering from activation of human neutrophils

    SciTech Connect

    Reibman, J.; Korchak, H.M.; Vosshall, L.B.; Haines, K.A.; Rich, A.M.; Weissmann, G.

    1988-05-05

    Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O/sub 2//sup -/) which are potent mediators of inflammation. Various agents elicit different responses. In contrast, phorbol myristate acetate (PMA, 1.6 ..mu..M) acting directly via protein kinase C is a potent stimulus for O/sub 2//sup -/. The authors compared the kinetics of appearance of various second messengers with the capacity of these ligands to elicit O/sub 2//sup -/ generation. Kinetic analysis showed a two-phase response to membrane ligands; both an early (greater than or equal to 15 s) and a late (>15 s) increase in (/sup 3/H)- and (/sup 14/C)diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB/sub 4/ elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Moreover, comparison of increases in (/sup 3/H)DG versus those of (/sup 14/C)DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 M/sub r/ protein was phosphorylated with kinetics consistent with the production of O/sub 2//sup -/ and DG in response to fMLP and PMA. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only triggering (the rapid generation of a signal) but also activation (the maintenance of a signal) to sustain responses.

  20. Deficiency of diacylglycerol kinase η induces lithium-sensitive mania-like behavior.

    PubMed

    Isozaki, Takeshi; Komenoi, Suguru; Lu, Qiang; Usuki, Takako; Tomokata, Shuntaro; Matsutomo, Daisuke; Sakai, Hiromichi; Bando, Kana; Kiyonari, Hiroshi; Sakane, Fumio

    2016-08-01

    The η isozyme of diacylglycerol kinase (DGK) is highly expressed in the hippocampus and Purkinje cells in the central nervous system. Recently, several genome-wide association studies have implicated DGKη in the etiology of bipolar disorder (BPD). However, it is still unknown whether DGKη is indeed related to BPD. In this study, we generated DGKη-knockout (KO) mice and performed behavioral tests such as the open field test, the elevated plus maze test and tail suspension test using the KO mice to investigate the effects of DGKη deficits on psychomotor behavior. Intriguingly, DGKη-KO mice displayed an overall behavioral profile that is similar to human mania, including hyperactivity, less anxiety and less depression-like behavior. In addition, these phenotypes were significantly attenuated by the administration of a BPD (mania) remedy, namely, lithium. Moreover, DGKη-KO mice showed impairment in glycogen synthase kinase (GSK) 3β signaling, which is closely related to BPD. These findings clearly support the linkage between BPD and DGKη that is implicated by genome-wide association studies. Moreover, this study provides DGKη-KO mice as a previously unrecognized model that reflects several features of human BPD with manic episodes and revealed an important role for DGKη in regulating behavior and mood through, at least in part, GSK3β signaling. Several genome-wide association studies have implicated diacylglycerol kinase (DGK) η gene in the etiology of bipolar disorder (BPD). In this study, we revealed that DGKη-knockout (KO) mice displayed an overall behavioral profile that is similar to mania of BPD and is lithium (BPD (mania) remedy)-sensitive. DGKη may regulate behavior and mood through, at least in part, glycogen synthase kinase (GSK) 3β signaling.

  1. Loss of Diacylglycerol Kinase-Ζ Inhibits Cell Proliferation and Survival in Human Gliomas.

    PubMed

    Diao, Jinfu; Wu, Chunyong; Zhang, Junying; Liu, Jialin; Zhang, Xinwu; Hao, Pengcheng; Zhao, Shanmin; Zhang, Zhiwen

    2016-10-01

    Diacylglycerol kinases ζ (DGKζ) is a critical lipid kinase which is involved in phosphatidic acid (PA) generation via diacylglycerol (DAG) phosphorylation. DGKζ is highly expressed in central nervous system and essential for brain development. Studies have indicated that DGKζ is associated with colon cancer invasion and metastasis. However, the involvement of DGKζ in human glioma development remains elusive. Here, we explored the impact and possible mechanisms of DGKζ knockdown on the proliferation and survival of glioma cells. The relationship between DGKζ expression status and human glioma stages was explored in 111 specimens of human gliomas via immunohistochemistry technology. Then the impact of DGKζ on cell proliferation, cell cycle, survival, and colony formation ability was determined in U-87 MG glioma cell lines via lentiviral-mediated small interfering (shRNA) strategy. The influence of DGKζ knockdown on global gene expression in U-87 MGglioma cell lines was further analyzed by microarray platform to reveal the possible molecular mechanisms underlying DGKζ-mediated glioma development and progression. Immunohistochemistry analysis revealed that DGKζ expression is positively correlated with human gliomagrade. Lentiviral-mediated small interfering (shRNA) strategyefficiently reduced DGKζ expression and DGKζ knockdown impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and cell apoptosis in U-87 MG glioma cells. Finally, microarray analysis revealed that multiple cancer-associated pathways and oncogenes were regulated by DGKζ knockdown, which provides insights into underlying mechansims of DGKζ-associated glioma development and progression. Our results established the positive correlation between DGKζ expression and gliomagrade. Furthermore, DGKζ knockdown in human glioma cell lines U-87 MG impaired cell proliferation, inhibited colony formation ability, and induced cell cycle arrest and apoptosis

  2. 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

  3. Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae.

    PubMed

    Tian, Siqi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-01-01

    To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

  4. 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

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

    PubMed

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

    2015-11-01

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

  6. Synthesis of diacylglycerol de novo is responsible for permanent activation and down-regulation of protein kinase C in transformed cells

    SciTech Connect

    Chiarugi, V.; Bruni, P.; Pasquali, F.; Magnelli, L.; Basi, G.; Ruggiero, M.; Farnararo, M. )

    1989-10-31

    We measured the synthesis of diacylglycerol de novo in normal NIH/3T3 fibroblasts and in cells transformed by ras, src, sis and abl oncogenes. Analysis of the incorporation of glucose-derived {sup 14}C into diacylglycerol indicated that neosynthesis of diacylglycerol was constitutively active in the transformed cell lines. Elevated levels of diacylglycerol and persistent activation/down-regulation of protein kinase C reduced the binding of phorbol dibutyrate to transformed cells. This phenomenon could be reversed by blocking the glycolytic pathway, thus indicating that neosynthesized diacylglycerol was responsible for persistent activation and down-regulation of protein kinase C. In transformed cells, protein kinase C activity could not be stimulated by the addition of diolein; however, inhibition of glycolysis restored the ability of transformed cells to respond to diolein. Taken together these data indicate that constitutive synthesis of diacylglycerol de novo is responsible for activation and down-regulation of protein kinase C in transformed cells, and it may play a role in altered mitogenic signalling.

  7. Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1.

    PubMed

    Harada, Bryan T; Knight, Mary Jane; Imai, Shin-Ichi; Qiao, Feng; Ramachander, Ranjini; Sawaya, Michael R; Gingery, Mari; Sakane, Fumio; Bowie, James U

    2008-03-01

    The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

  8. Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases

    SciTech Connect

    Miller, Darcie J.; Jerga, Agoston; Rock, Charles O.; White, Stephen W.

    2008-08-11

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg{sup 2+} site and an associated Asp{center_dot}water{center_dot}Mg{sup 2+} network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp{center_dot}water{center_dot}Mg{sup 2+} network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  9. Tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase

    SciTech Connect

    Mentlein, R.

    1986-06-15

    Rat liver homogenate or cell fractions deacylate 12-O-tetradecanoyl phorbol 13-acetate (TPA) in vitro mainly by conversion to phorbol 13-acetate. The highest specific activity is located in the microsomal fraction. The deacylation is inhibited by bis-(4-nitrophenyl) phosphate, a selective inhibitor of nonspecific carboxylesterases. Only two of five purified esterases from rat liver endoplasmic reticulum deacylate TPA. These two esterases have formerly been characterized as acylcarnitine hydrolases and the more active one is also a potent diacylglycerol lipase. Its TPA-hydrolyzing activity is inhibited by other substrates like 1-naphthylacetate, lauroylcarnitine, or dioleoyl glycerol. The results support the view that phorbol esters act like structural analogs of diacylglycerols, not only with respect to their activating effect on protein kinase C, but also as substrates for the same lipases.

  10. Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1.

    PubMed

    Ruffo, Elisa; Malacarne, Valeria; Larsen, Sasha E; Das, Rupali; Patrussi, Laura; Wülfing, Christoph; Biskup, Christoph; Kapnick, Senta M; Verbist, Katherine; Tedrick, Paige; Schwartzberg, Pamela L; Baldari, Cosima T; Rubio, Ignacio; Nichols, Kim E; Snow, Andrew L; Baldanzi, Gianluca; Graziani, Andrea

    2016-01-13

    X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.

  11. Mechanism for release of arachidonic acid during guinea pig platelet aggregation: a role for the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.

    1986-01-01

    The mechanism of the release of arachidonic acid from phospholipids after the stimulation of guinea pig platelets with collagen, thrombin and platelet activating factor (PAF) was studied. RHC 80267, a diacylglycerol lipase inhibitor, and indomethacin, a cyclooxygenase inhibitor, were used. Various in vitro assays for enzymes involved in arachidonic acid release and metabolism were conducted. Platelet aggregation and simultaneous release of ADP from platelets were monitored using a Chrono-log Lumiaggregometer. Platelets were labeled with (/sup 14/C)arachidonic acid to facilitate sensitive determination of small changes in platelet phospholipids during platelet aggregation. In the present investigation it is shown that collagen, thrombin and PAF increased phospholipase C activity. It was also discovered that cyclooxygenase products were responsible for further stimulation (a positive feed-back) of phospholipase C activity, while diacylglycerol provided a negative feed-back control over receptor-stimulated phospholipase C activity and inhibited ADP release. The guinea pig platelet is an ideal model to study phospholipase C-diacylglycerol lipase pathway for the release of arachidonic acid from platelet phospholipids because it does not have any phospholipase A/sub 2/ activity. It was observed that cyclooxygenase products were responsible for collagen-induced guinea pig platelet aggregation. Indomethacin completely inhibited collagen-induced platelet aggregation, was less effective against thrombin, and had no effect on PAF-induced platelet aggregation. On the other hand, RHC 80267 was a powerful inhibitor of aggregation and ADP release induced by all three of these potent aggregating agents.

  12. 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

  13. A Fluorescence Method to Detect and Quantitate Sterol Esterification by Lecithin: Cholesterol Acyltransferase

    PubMed Central

    Homan, Reynold; Esmaeil, Nadia; Mendelsohn, Laurel; Kato, Gregory J.

    2013-01-01

    We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity. PMID:23851343

  14. Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.

    PubMed

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

    2013-10-28

    Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.

  15. Catalytic properties of alcohol acyltransferase in different strawberry species and cultivars.

    PubMed

    Olías, Raquel; Pérez, Ana G; Sanz, Carlos

    2002-07-01

    The substrate specificity of alcohol acyltransferase (AAT) enzymes from different strawberry varieties was studied. Proteins with AAT activity from fruits of Fragaria x ananassa Duch. cv. Oso Grande were purified to apparent homogeneity and used for kinetic studies with different straight-chain alcohols and acyl-CoAs. K(m) values obtained for Oso Grande enzyme with six different alcohols, using acetyl-CoA as cosubstrate, decreased with increasing length of the alcohol chain. In similar experiments the increase in the acyl-CoA carbon chain was also found to be correlated with a higher substrate specificity. Heptanol (K(m) = 0.73 mM) and hexanoyl-CoA (K(m) = 0.41 mM) were the best substrates for Oso Grande AAT. Comparative catalytic studies were carried out with AAT partially purified extracts from the wild type Fragaria vesca and five commercial strawberry varieties: Tudnew, Carisma, Camarosa, Sweet Charlie, and Eris. The specificities of these enzymes toward five selected alcohols and acyl-CoAs reflected interesting cultivar differences.

  16. 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

  17. 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.

  18. Roles of cysteine 161 and tyrosine 154 in the lecithin-retinol acyltransferase mechanism.

    PubMed

    Xue, Linlong; Rando, Robert R

    2004-05-25

    Lecithin-retinol acyltransferase (LRAT) catalyzes the transfer of an acyl moiety from the sn-1 position of lecithin to vitamin A, generating all-trans-retinyl esters. LRAT is a unique enzyme and is the founder member of an expanding group of proteins of largely unknown function. In an effort to understand the mechanism of LRAT action, it was of interest to assign the amino acid residues responsible for the two pK(a) values of 8.22 and 9.95 observed in the pH vs rate profile. Titrating C161 of LRAT with a specific affinity labeling agent at varying pH values shows that this residue has a pK(a) = 8.03. Coupled with previous studies, this titration reveals the catalytically essential C161 as the residue responsible for the ascending limb of the pH vs rate profile. Site-specific mutagenic experiments on the lysine and tyrosine residues of LRAT reveal that only the highly conserved tyrosine 154 is essential for catalytic activity. This residue is likely to be responsible for the pK(a) = 9.95 found in the pH vs rate profile. Thus, LRAT has three essential residues (C161, Y154, and H60), all of which are conserved in the LRAT family of enzymes.

  19. Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism.

    PubMed

    Hoffmann, Laurent; Maury, Stephane; Martz, Francoise; Geoffroy, Pierrette; Legrand, Michel

    2003-01-01

    A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.

  20. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by 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

    2015-12-01

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

  1. Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome

    PubMed Central

    Beard, Richard S.; Yang, Xiaoyuan; Meegan, Jamie E.; Overstreet, Jonathan W.; Yang, Clement G.Y.; Elliott, John A.; Reynolds, Jason J.; Cha, Byeong J.; Pivetti, Christopher D.; Mitchell, David A.; Wu, Mack H.; Deschenes, Robert J.; Yuan, Sarah Y.

    2016-01-01

    Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCβ1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCβ1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCβ1 palmitoylation in endothelial inflammation. PMID:27653213

  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. AM-251 and SR144528 are acyl CoA:cholesterol acyltransferase inhibitors

    PubMed Central

    Thewke, Douglas; Freeman-Anderson, Natalie; Pickle, Theresa; Netherland, Courtney; Chilton, Courtney

    2009-01-01

    Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2 +/+ and CB2−/− peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have anti-atherosclerotic activities independent of their affect on cannabinoid signaling. PMID:19338772

  4. 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

  5. JTP-103237, a novel monoacylglycerol acyltransferase inhibitor, modulates fat absorption and prevents diet-induced obesity.

    PubMed

    Okuma, Chihiro; Ohta, Takeshi; Tadaki, Hironobu; Hamada, Hiromi; Oda, Tomohiro; Taniuchi, Hideyuki; Yamanaka, Kenji; Ishii, Yukihito; Ohe, Yasuhiro; Yata, Shinji; Nishiu, Jun; Aratsu, Yusuke; Oshida, Shinichi; Kume, Shinichi; Kakutani, Makoto

    2015-07-01

    Monoacylglycerol acyltransferase 2 (MGAT2) plays an important role in intestinal fat absorption. We discovered the novel MGAT2 inhibitor, JTP-103237, and evaluated its pharmacological profile. JTP-103237 selectively inhibited MGAT2 without remarkable species differences and reduced absorbed lipids in circulation. After lipid administration, JTP-103237 slightly but significantly decreased triglyceride content in proximal small intestine and significantly increased the lipids content in the distal small intestine. In addition, JTP-103237 significantly increased MGAT substrate (monoacylglycerol and fatty acid) content in the small intestine. JTP-103237 increased plasma peptide YY levels after lipid loading and reduced food intake in a dietary fat-dependent manner. After chronic treatment, JTP-103237 significantly decreased body weight and increased O2 consumption in the early dark phase in high fat diet induced obese (DIO) mice. Moreover, JTP-103237 improved glucose tolerance and decreased fat weight and hepatic triglyceride content in DIO mice. Our findings indicate that JTP-103237 prevents diet-induced obesity by inhibiting intestinal MGAT2 and has unique properties as a drug for the treatment of obesity.

  6. 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.

  7. Homeostasis of brassinosteroids regulated by DRL1, a putative acyltransferase in Arabidopsis.

    PubMed

    Zhu, Wenjiao; Wang, Haijiao; Fujioka, Shozo; Zhou, Tao; Tian, Hailong; Tian, Weisheng; Wang, Xuelu

    2013-03-01

    Brassinosteroids (BRs) play essential roles in regulating various aspects of plant growth and development and in responding to diverse environmental cues, and their metabolism is an important way to regulate their homeostasis in plants. Here, we identified a dominant mutant, dwarf and round leaf-1 (drl1-D), which exhibits weak BR-deficient or BR-insensitive mutant phenotypes, including short and round leaves, prolonged senescence, dwarfed shape, and altered expression levels of the BR-responsive genes. Hypocotyl length and root inhibition assays suggest that the drl1-D mutant responds to BRs normally, but has decreased BR signaling outputs. The endogenous levels of several BRs, including typhasterol (TY), 6-deoxotyphasterol (6-deoxoTY), and 6-deoxocastasterone (6-deoxoCS), are significantly lower in the drl1-D mutant than in the wild-type. The DRL1 gene encodes an acyltransferase and is widely expressed in leaves, roots, flowers, and siliques. Plants without DRL1 and its homologs are larger with an enhanced BR signaling. The expression of DRL1 was induced by eBL and inhibited by ABA. DRL1 is involved in the BR metabolism likely by catalyzing the BR conjugation through esterification, which plays important roles in regulating the BR homeostasis and responding to abiotic stresses in Arabidopsis. PMID:23204503

  8. Sequence-specific apolipoprotein A-I effects on lecithin:cholesterol acyltransferase activity.

    PubMed

    Dergunov, Alexander D

    2013-06-01

    Existing kinetic data of cholesteryl ester formation by lecithin:cholesterol acyltransferase in discoidal high-density lipoproteins with 34 mutations of apoA-I that involved all putative helices were grouped by cluster analysis into four noncoincident regions with mutations both without any functional impairment and with profound isolated (V- and K-mutations) or common (VK-mutations) effect on V(max)(app) and K(m)(app). Data were analyzed with a new kinetic model of LCAT activity at interface that exploits the efficiency of LCAT binding to the particle, particle dimensions, and surface concentrations of phosphatidylcholine and cholesterol. V-mutations with major location in the central part and C-domain affected the second-order rate constant of cholesteryl ester formation at the solvolysis of acyl-enzyme intermediate by cholesterol as nucleophile. The central region in apoA-I sequence is suggested to influence the proper positioning of cholesterol molecule toward LCAT active center with major contribution of arginine residue(s). K-mutations with major location in N-domain may affect binding and stability of enzyme-phosphatidylcholine complex. VK-mutations may possess mixed effects; the independent binding measurement may segregate individual steps. PMID:23516040

  9. 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

  10. A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase.

    PubMed

    Homan, Reynold; Esmaeil, Nadia; Mendelsohn, Laurel; Kato, Gregory J

    2013-10-01

    We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity. PMID:23851343

  11. 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

  12. 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

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

    PubMed

    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; Brown, J Mark; Temel, Ryan E

    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.

  14. 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

  15. 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

  16. 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

  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.

  18. Homeostasis of brassinosteroids regulated by DRL1, a putative acyltransferase in Arabidopsis.

    PubMed

    Zhu, Wenjiao; Wang, Haijiao; Fujioka, Shozo; Zhou, Tao; Tian, Hailong; Tian, Weisheng; Wang, Xuelu

    2013-03-01

    Brassinosteroids (BRs) play essential roles in regulating various aspects of plant growth and development and in responding to diverse environmental cues, and their metabolism is an important way to regulate their homeostasis in plants. Here, we identified a dominant mutant, dwarf and round leaf-1 (drl1-D), which exhibits weak BR-deficient or BR-insensitive mutant phenotypes, including short and round leaves, prolonged senescence, dwarfed shape, and altered expression levels of the BR-responsive genes. Hypocotyl length and root inhibition assays suggest that the drl1-D mutant responds to BRs normally, but has decreased BR signaling outputs. The endogenous levels of several BRs, including typhasterol (TY), 6-deoxotyphasterol (6-deoxoTY), and 6-deoxocastasterone (6-deoxoCS), are significantly lower in the drl1-D mutant than in the wild-type. The DRL1 gene encodes an acyltransferase and is widely expressed in leaves, roots, flowers, and siliques. Plants without DRL1 and its homologs are larger with an enhanced BR signaling. The expression of DRL1 was induced by eBL and inhibited by ABA. DRL1 is involved in the BR metabolism likely by catalyzing the BR conjugation through esterification, which plays important roles in regulating the BR homeostasis and responding to abiotic stresses in Arabidopsis.

  19. 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

  20. P2Y₁ receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion.

    PubMed

    Wuttke, Anne; Idevall-Hagren, Olof; Tengholm, Anders

    2013-04-01

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes.

  1. Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4.

    PubMed

    Murakami, Eri; Shionoya, Takao; Komenoi, Suguru; Suzuki, Yuji; Sakane, Fumio

    2016-01-01

    Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner. PMID:27643686

  2. Does Diacylglycerol Accumulation in Fatty Liver Disease Cause Hepatic Insulin Resistance?

    PubMed Central

    Finck, Brian N.; Hall, Angela M.

    2015-01-01

    Numerous studies conducted on obese humans and various rodent models of obesity have identified a correlation between hepatic lipid content and the development of insulin resistance in liver and other tissues. Despite a large body of the literature on this topic, the cause and effect relationship between hepatic steatosis and insulin resistance remains controversial. If, as many believe, lipid aggregation in liver drives insulin resistance and other metabolic abnormalities, there are significant unanswered questions as to which lipid mediators are causative in this cascade. Several published papers have now correlated levels of diacylglycerol (DAG), the penultimate intermediate in triglyceride synthesis, with development of insulin resistance and have postulated that this occurs via activation of protein kinase C signaling. Although many studies have confirmed this relationship, many others have reported a disconnect between DAG content and insulin resistance. It has been postulated that differences in methods for DAG measurement, DAG compartmentalization within the cell, or fatty acid composition of the DAG may explain these discrepancies. The purpose of this review is to compare and contrast some of the relevant findings in this area and to discuss a number of unanswered questions regarding the relationship between DAG and insulin resistance. PMID:26273583

  3. Identification of small molecules that selectively inhibit diacylglycerol lipase-α activity.

    PubMed

    Appiah, Kingsley K; Blat, Yuval; Robertson, Barbara J; Pearce, Bradley C; Pedicord, Donna L; Gentles, Robert G; Yu, Xuan-Chuan; Mseeh, Faika; Nguyen, Nghi; Swaffield, Jonathan C; Harden, David G; Westphal, Ryan S; Banks, Martyn N; O'Connell, Jonathan C

    2014-04-01

    Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization. PMID:24241710

  4. Determination of physicochemical properties of diacylglycerol oil at high pressure by means of ultrasonic methods.

    PubMed

    Kiełczyński, Piotr; Szalewski, Marek; Balcerzak, Andrzej; Wieja, Krzysztof; Malanowski, Aleksander; Kościesza, Rafał; Tarakowski, Rafał; Rostocki, Aleksander J; Siegoczyński, Ryszard M

    2014-12-01

    The purpose of the paper is to address, using ultrasonic methods, the impact of temperature and pressure on the physicochemical properties of liquids on the example of diacylglycerol (DAG) oil. The paper presents measurements of sound velocity, density and volume of DAG oil sample in the pressure range from atmospheric pressure up to 0.6GPa and at temperatures ranging from 20 to 50°C. Sound speed measurements were performed in an ultrasonic setup with a DAG oil sample located in the high-pressure chamber. An ultrasonic method that uses cross-correlation method to determine the time-of-flight of the ultrasonic pulses through the liquid was employed to measure the sound velocity in DAG oil. This method is fast and reliable tool for measuring sound velocity. The DAG oil density at high pressure was determined from the monitoring of sample volume change. The adiabatic compressibility and isothermal compressibility have been calculated on the basis of experimental data. Discontinuities in isotherms of the sound speed versus pressure point to the existence of phase transitions in DAG oil. The ultrasonic method presented in this study can be applied to investigate the physicochemical parameters of other liquids not only edible oils. PMID:25017363

  5. Potential role of salicylic acid in modulating diacylglycerol homeostasis in response to freezing temperatures in Arabidopsis.

    PubMed

    Tan, Wei-Juan; Xiao, Shi; Chen, Qin-Fang

    2015-01-01

    In our recent article in Molecular Plant, we reported that 3 lipase-like defense regulators SENESCENCE-ASSOCIATED GENE101 (SAG101), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are involved in the regulation of freezing tolerance in Arabidopsis. The transcripts of SAG101, EDS1 and PAD4 were inducible by cold stress and their knockout or knockdown mutants exhibited enhanced chilling and freezing tolerance in comparison to the wild type. The freezing tolerance phenotype showed in the sag101, eds1 and pad4 mutants was correlated with the transcriptional upregulation of C-REPEAT/DRE BINDING FACTORs (CBFs) and their regulons as well as increased levels of proline. Upon cold exposure, the sag101, eds1 and pad4 mutants showed ameliorated cell death and accumulation of hydrogen peroxide, which were highly induced by freezing stress in the wild-type leaves. Moreover, the contents of salicylic acid (SA) and diacylglycerol (DAG) were significantly decreased in the sag101, eds1 and pad4 mutants compared to the wild type. Taken together, our results suggest that the SAG101, EDS1 and PAD4 are negative regulators in the freezing response and function, at least in part, by modulating the homeostasis of SA and DAG in Arabidopsis.

  6. CaMKII regulates diacylglycerol lipase-α and striatal endocannabinoid signaling.

    PubMed

    Shonesy, Brian C; Wang, Xiaohan; Rose, Kristie L; Ramikie, Teniel S; Cavener, Victoria S; Rentz, Tyler; Baucum, Anthony J; Jalan-Sakrikar, Nidhi; Mackie, Ken; Winder, Danny G; Patel, Sachin; Colbran, Roger J

    2013-04-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the molecular regulation of 2-AG synthesis is not well understood. Here we identify a functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in 2-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of 2-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of 2-AG breakdown using concentrations of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the molecular regulation of striatal endocannabinoid signaling with implications for physiological control of motor function.

  7. Determination of physicochemical properties of diacylglycerol oil at high pressure by means of ultrasonic methods.

    PubMed

    Kiełczyński, Piotr; Szalewski, Marek; Balcerzak, Andrzej; Wieja, Krzysztof; Malanowski, Aleksander; Kościesza, Rafał; Tarakowski, Rafał; Rostocki, Aleksander J; Siegoczyński, Ryszard M

    2014-12-01

    The purpose of the paper is to address, using ultrasonic methods, the impact of temperature and pressure on the physicochemical properties of liquids on the example of diacylglycerol (DAG) oil. The paper presents measurements of sound velocity, density and volume of DAG oil sample in the pressure range from atmospheric pressure up to 0.6GPa and at temperatures ranging from 20 to 50°C. Sound speed measurements were performed in an ultrasonic setup with a DAG oil sample located in the high-pressure chamber. An ultrasonic method that uses cross-correlation method to determine the time-of-flight of the ultrasonic pulses through the liquid was employed to measure the sound velocity in DAG oil. This method is fast and reliable tool for measuring sound velocity. The DAG oil density at high pressure was determined from the monitoring of sample volume change. The adiabatic compressibility and isothermal compressibility have been calculated on the basis of experimental data. Discontinuities in isotherms of the sound speed versus pressure point to the existence of phase transitions in DAG oil. The ultrasonic method presented in this study can be applied to investigate the physicochemical parameters of other liquids not only edible oils.

  8. Cloning and Characterization of Novel Testis-Specific Diacylglycerol Kinase η Splice Variants 3 and 4

    PubMed Central

    Murakami, Eri; Shionoya, Takao; Komenoi, Suguru; Suzuki, Yuji; Sakane, Fumio

    2016-01-01

    Diacylglycerol kinase (DGK) phosphorylates DG to generate phosphatidic acid. Recently, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the secondary spermatocytes and round spermatids of the testis. In this study, we cloned the full length DGKη3 gene and confirmed the endogenous expression of its protein product. During the cloning procedure, we found a new testis-specific alternative splicing product of the DGKη gene, DGKη4, which lacks half of the catalytic domain. We examined the DGK activity and subcellular localization of DGKη3 and η4. DGKη3 had almost the same activity as DGKη1, whereas the activity of DGKη4 was not detectable. In resting NEC8 cells (human testicular germ cell tumor cell line), DGKη1, η3 and η4 were broadly distributed in the cytoplasm. When osmotically shocked, DGKη1 and η4 were distributed in punctate vesicles in the cytoplasm. In contrast, DGKη3 was partly translocated to the plasma membrane and co-localized with the actin cytoskeleton. These results suggest that DGKη3 and η4 have properties different from those of DGKη1 and that they play roles in the testis in a different manner. PMID:27643686

  9. Diacylglycerol Kinases as Emerging Potential Drug Targets for a Variety of Diseases: An Update

    PubMed Central

    Sakane, Fumio; Mizuno, Satoru; Komenoi, Suguru

    2016-01-01

    Ten mammalian diacylglycerol kinase (DGK) isozymes (α–κ) have been identified to date. Our previous review noted that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes (Sakane et al., 2008). Since then, recent genome-wide association studies have implied several new possible relationships between DGK isozymes and diseases. For example, DGKθ and DGKκ have been suggested to be associated with susceptibility to Parkinson's disease and hypospadias, respectively. In addition, the DGKη gene has been repeatedly identified as a bipolar disorder (BPD) susceptibility gene. Intriguingly, we found that DGKη-knockout mice showed lithium (BPD remedy)-sensitive mania-like behaviors, suggesting that DGKη is one of key enzymes of the etiology of BPD. Because DGKs are potential drug targets for a wide variety of diseases, the development of DGK isozyme-specific inhibitors/activators has been eagerly awaited. Recently, we have identified DGKα-selective inhibitors. Because DGKα has both pro-tumoral and anti-immunogenic properties, the DGKα-selective inhibitors would simultaneously have anti-tumoral and pro-immunogenic (anti-tumor immunogenic) effects. Although the ten DGK isozymes are highly similar to each other, our current results have encouraged us to identify and develop specific inhibitors/activators against every DGK isozyme that can be effective regulators and drugs against a wide variety of physiological events and diseases. PMID:27583247

  10. Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development

    PubMed Central

    Shen, Shudan; Wu, Jinhong; Srivatsan, Sruti; Gorentla, Balachandra; Shin, Jinwook; Xu, Li; Zhong, Xiao-Ping

    2011-01-01

    Type I natural killer T (NKT) cells, or iNKT cells, express a semi-invariant T cell receptor characterized by its unique V α 14-Jα 18 usage (iV α 14TCR). Upon interaction with glycolipid/CD1d complexes, the iV α 14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the PKCθ-IKKα/β-NFκB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases (DGKs) play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. Here we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NFκB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development. PMID:21775687

  11. Diacylglycerol kinase-δ regulates AMPK signaling, lipid metabolism, and skeletal muscle energetics.

    PubMed

    Jiang, Lake Q; de Castro Barbosa, Thais; Massart, Julie; Deshmukh, Atul S; Löfgren, Lars; Duque-Guimaraes, Daniella E; Ozilgen, Arda; Osler, Megan E; Chibalin, Alexander V; Zierath, Juleen R

    2016-01-01

    Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase-δ (DGKδ), an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here, we tested the hypothesis that DGKδ plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGKδ(+/-) mice, but glycogen content and mitochondrial markers were unaltered, suggesting that DGKδ deficiency affects skeletal muscle energetics but not mitochondrial protein abundance. We next determined the role of DGKδ in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling were reduced in DGKδ(+/-) mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGKδ deficiency impaired work performance, as evident by altered force production and relaxation dynamics in response to repeated contractions. In conclusion, DGKδ deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGKδ deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.

  12. Myristic Acid Enhances Diacylglycerol Kinase δ-Dependent Glucose Uptake in Myotubes.

    PubMed

    Wada, Yuko; Sakiyama, Shizuka; Sakai, Hiromichi; Sakane, Fumio

    2016-08-01

    Decreased expression of diacylglycerol kinase (DGK) δ in skeletal muscles attenuates glucose uptake and is closely related to the pathogenesis of type 2 diabetes. Therefore, up-regulation of DGKδ expression is thought to protect and improve glucose homoeostasis in type 2 diabetes. We recently determined that myristic acid (14:0), but not palmitic (16:0) or stearic (18:0) acid, significantly increased DGKδ2 protein expression in mouse C2C12 myotubes. In the current study, we analyzed whether myristic acid indeed enhances glucose uptake in C2C12 myotubes. We observed that myristic acid caused ~1.4-fold increase in insulin-independent glucose uptake. However, palmitic and stearic acids failed to enhance glucose uptake. DGKδ-specific siRNA decreased myristic acid-dependent increase of glucose uptake. Moreover, overexpression of DGKδ2 enhanced glucose uptake in C2C12 cells in the absence of myristic acid treatment. Taken together, these results strongly suggest that myristic acid enhances basal glucose uptake in myotubes in a DGKδ2 expression-dependent manner.

  13. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

  14. Angiotensin II increases diacylglycerol in calf adrenal glomerulosa cells by activating de novo phospholipid synthesis

    SciTech Connect

    Foster, R.H.; Farese, R.V. )

    1989-01-01

    Effects of angiotension II (AII) on diacylglycerol (DAG) synthesis were examined in calf adrenal glomerulosa cells. AII provoked rapid increases in ({sup 3}H) glycerol-labeling and content of DAG. Effects on ({sup 3}H) glycerol-labeling of DAG were observed both in cells prelabeled with ({sup 3}H) glycerol for 60 minutes, and when AII and ({sup 3}H) glycerol were added simultaneously. Increases in ({sup 3}H) DAG labeling were associated with increases in total glycerolipid labeling, and in simultaneous addition experiments, were preceded by increased ({sup 3}H) phosphatidic acid (PA) labeling. Labeling of glycerol-3-PO{sub 4}, on the other hand, was not increased by AII, suggesting that increases in lipid labeling were not due to prior increases in precursor specific activity. ACTH, which were not increase precursor specific activity. ACTH, which does not increase the hydrolysis of inositol-phospholipids appreciably in this tissue, provoked increases in content and ({sup 3}H) glycerol-labeling of DAG, which were only slightly less than those provoked by AII. Thus, part of the AII-induced increase in DAG may also be derived from sources other than inositol-phospholipids. Moreover, AII-induced increase in DAG appear to be at least partly derived from increased de novo synthesis of PA.

  15. Evidence for a requirement of agonist-induced diacylglycerol production during tonic contraction of rat aorta

    SciTech Connect

    Not Available

    1986-03-01

    A possible role for protein kinase C during the tonic phase of arterial contraction was examined in rat aorta by observing the effects of the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), on angiotensin II (AII)-induced responses. The ability of AII and phenylephrine (PE) to induce diacylglycerol (DAG) production was monitored as agonist-stimulated /sup 32/P-labelling of phosphatidic acid (PA). AII (5 x 10/sup -7/M) causes only a transient contractile response, while PE (10/sup -5/M) causes a sustained tonic contraction. /sup 32/P-labelling studies showed that AII caused an initial increase of PA synthesis equal to PE, however, AII failed to sustain this increase at 5 and 10 min while PE was able to do so, indicating the failure of AII to provide DAG to sustain protein kinase C activation. Activation of protein kinase C with TPA prior to and during AII exposure converted the normally transient contraction to a more sustained, tonic pattern. These results suggest that the inability of AII to maintain tension, unlike PE, is due to its inability to produce DAG continuously and activate protein kinase C.

  16. Production of extremely pure diacylglycerol from soybean oil by lipase-catalyzed glycerolysis.

    PubMed

    Wang, Weifei; Li, Tie; Ning, Zhengxiang; Wang, Yonghua; Yang, Bo; Yang, Xiaoquan

    2011-07-10

    Research work was objectively targeted to synthesize highly pure diacylglycerol (DAG) with glycerolysis of soybean oil in a solvent medium of t-butanol. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best out of three candidates. Batch reaction conditions of the enzymatic glycerolysis, the substrate mass ratio, the reaction temperature and the substrate concentration, were studied. The optimal reaction conditions were achieved as 6.23:1 mass ratio of soybean oil to glycerol, 40% (w/v) of substrate concentration in t-butanol and reaction temperature of 50 °C. A two-stage molecular distillation was employed for purification of DAG from reaction products. Scale-up was attempted based on the optimized reaction conditions, 98.7% (24 h) for the conversion rate of soybean oil, 48.5% of DAG in the glycerolysis products and 96.1% for the content of DAG in the final products were taken in account as the results.

  17. Process optimization of enzyme catalyzed production of dietary diacylglycerol (DAG) using TLIM as biocatalyst.

    PubMed

    Dhara, Rupali; Singhal, Rekha S

    2014-01-01

    Diacylglycerol (DAG)-rich sunflower oil was prepared and the optimal conditions for synthesis of DAG-rich oil by glycerolysis using biocatalyst TLIM was determined. A maximum production of 59.8% DAG was obtained after 5 h of constant reaction under vacuum (756 mm of Hg). The optimum temperature for glycerolysis was found to be 50°C, while stoichiometric molar ratio of sunflower oil:glycerol was 2:1 for this reaction. A minimum acid value of 0.48 mg of KOH.g(-1) of oil was observed under these conditions. The fatty acid composition of DAG-rich oil was found to be similar to the original TAG-rich sunflower oil used in the work. The lipase catalysed glycerolysis using 1,3 specific lipase was used to promote the formation of 1,3 isoform of DAG as this isoform is known to possess anti-obesity effect. DAG content was determined by HPTLC and GCMS. The DAG-rich oil contained 59.75% DAG of which 63.34% was found as 1,3-DAG and 36.65% was 1,2-DAG/2,3-DAG.

  18. Diacylglycerol Kinases as Emerging Potential Drug Targets for a Variety of Diseases: An Update.

    PubMed

    Sakane, Fumio; Mizuno, Satoru; Komenoi, Suguru

    2016-01-01

    Ten mammalian diacylglycerol kinase (DGK) isozymes (α-κ) have been identified to date. Our previous review noted that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes (Sakane et al., 2008). Since then, recent genome-wide association studies have implied several new possible relationships between DGK isozymes and diseases. For example, DGKθ and DGKκ have been suggested to be associated with susceptibility to Parkinson's disease and hypospadias, respectively. In addition, the DGKη gene has been repeatedly identified as a bipolar disorder (BPD) susceptibility gene. Intriguingly, we found that DGKη-knockout mice showed lithium (BPD remedy)-sensitive mania-like behaviors, suggesting that DGKη is one of key enzymes of the etiology of BPD. Because DGKs are potential drug targets for a wide variety of diseases, the development of DGK isozyme-specific inhibitors/activators has been eagerly awaited. Recently, we have identified DGKα-selective inhibitors. Because DGKα has both pro-tumoral and anti-immunogenic properties, the DGKα-selective inhibitors would simultaneously have anti-tumoral and pro-immunogenic (anti-tumor immunogenic) effects. Although the ten DGK isozymes are highly similar to each other, our current results have encouraged us to identify and develop specific inhibitors/activators against every DGK isozyme that can be effective regulators and drugs against a wide variety of physiological events and diseases. PMID:27583247

  19. Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for Histidine Stabilization of the Malonyl-Enzyme Intermediate

    PubMed Central

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

    2014-01-01

    Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. 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. PMID:25286165

  20. 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.

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

    DOE PAGES

    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

  2. The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase

    PubMed Central

    Knight, Michael J; Bull, Ian D; Curnow, Paul

    2014-01-01

    Fatty acid ethyl esters are secondary metabolites that are produced during microbial fermentation, in fruiting plants and in higher organisms during ethanol stress. In particular, volatile medium-chain fatty acid ethyl esters are important flavour compounds that impart desirable fruit aromas to fermented beverages, including beer and wine. The biochemical synthesis of medium-chain fatty acid ethyl esters is poorly understood but likely involves acyl-CoA:ethanol O-acyltransferases. Here, we characterize the enzyme ethanol hexanoyl transferase 1 (Eht1) from the brewer's yeast Saccharomyces cerevisiae. Full-length Eht1 was successfully overexpressed from a recombinant yeast plasmid and purified at the milligram scale after detergent solubilization of sedimenting membranes. Recombinant Eht1 was functional as an acyltransferase and, unexpectedly, was optimally active toward octanoyl-CoA, with kcat = 0.28 ± 0.02/s and KM = 1.9 ± 0.6 μm. Eht1 was also revealed to be active as a thioesterase but was not able to hydrolyse p-nitrophenyl acyl esters, in contrast to the findings of a previous study. Low-resolution structural data and site-directed mutagenesis provide experimental support for a predicted α/β-hydrolase domain featuring a Ser–Asp–His catalytic triad. The S. cerevisiae gene YBR177C/EHT1 should thus be reannotated as coding for an octanoyl-CoA:ethanol acyltransferase that can also function as a thioesterase. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:25308280

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

    PubMed

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

    2012-09-14

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

  4. 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

  5. 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.

  6. INHIBITION OF GHRELIN O-ACYLTRANSFERASE ATTENUATES FOOD DEPRIVATION-INDUCED INCREASES IN INGESTIVE BEHAVIOR1

    PubMed Central

    Teubner, Brett J.W.; Garretson, John T.; Hwang, Yousang; Cole, Philip A.; Bartness, Timothy J.

    2013-01-01

    Ghrelin is an orexigenic hormone produced by the stomach in direct proportion to the time since the last meal and has therefore been called a ‘hunger signal’. The octanoylation of ghrelin is critical for its orexigenic functions and is dependent upon ghrelin O-acyltransferase (GOAT) catalyzation. The GOAT inhibitor, GO-CoA-Tat, decreases the circulating concentrations of octanoylated ghrelin and attenuates weight gain on a high fat diet in mice. Unlike rats and mice, Siberian hamsters and humans do not increase food intake after food deprivation, but increase food hoarding after food deprivation. In Siberian hamsters, exogenous ghrelin increases ingestive behaviors similarly to 48–56 h food deprivation. Therefore, we tested the necessity of increased ghrelin in food-deprived Siberian hamsters to stimulate ingestive behaviors. To do so we used our simulated natural housing system that allows hamsters to forage for and hoard food. Animals were given an injection of GO-CoA-Tat (i.p., 11 μmol/kg) every 6 h because that is the duration of its effective inhibition of octanoylated ghrelin concentrations during a 48 h food deprivation. We found that GO-CoA-Tat attenuated food foraging (0–1 h), food intake (0–1 and 2–4 h), and food hoarding (0–1 h and 2 and 3 d) post-refeeding compared with saline treated animals. This suggests that increased octanoylated ghrelin concentrations play a role in the food deprivation-induced increases in ingestive behavior. Therefore, ghrelin is a critical aspect of the multi-faceted mechanisms that stimulate ingestive behaviors, and might be a critical point for a successful clinical intervention scheme in humans. PMID:23399323

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

    PubMed Central

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

    2014-01-01

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

  8. 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

  9. High lysophosphatidylcholine acyltransferase 1 expression independently predicts high risk for biochemical recurrence in prostate cancers.

    PubMed

    Grupp, Katharina; Sanader, Stella; Sirma, Hüseyin; Simon, Ronald; Koop, Christina; Prien, Kristina; Hube-Magg, Claudia; Salomon, Georg; Graefen, Markus; Heinzer, Hans; Minner, Sarah; Izbicki, Jakob R; Sauter, Guido; Schlomm, Thorsten; Tsourlakis, Maria Christina

    2013-12-01

    Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been suggested to play a role in cancer. To assess its role in prostate cancer, LPCAT1 expression was analyzed on a tissue microarray containing samples from 11,152 prostate cancer patients. In benign prostate glands, LPCAT1 immunostaining was absent or weak. In prostate cancer, LPCAT1 positivity was found in 73.8% of 8786 interpretable tumors including 29.2% with strong expression. Increased LPCAT1 expression was associated with advanced tumor stage (pT3b/T4) (p < 0.0001), high Gleason score (≥4 + 4) (p < 0.0001), positive nodal involvement (p = 0.0002), positive surgical margin (p = 0.0005), and early PSA recurrence (p < 0.0001). High LPCAT1 expression was strongly linked to ERG-fusion type prostate cancer. Strong LPCAT1 staining was detected in 45.3% of ERG positive but in only 16.7% of ERG negative tumors (p < 0.0001). Within ERG negative cancers, LPCAT1 staining was strongly increased within the subgroup of PTEN deleted cancers (p < 0.0001). Further subgroup analyses revealed that associations of high LPCAT1 expression with PSA recurrence and unfavorable tumor phenotype were largely driven by ERG negative cancers (p < 0.0001) while these effects were substantially mitigated in ERG positive cancers (p = 0.0073). The prognostic impact of LPCAT1 expression was independent of histological and clinical parameters. It is concluded, that LPCAT1 measurement, either alone or in combination, may be utilized for better clinical decision-making. These data also highlight the potentially important role of lipid metabolism in prostate cancer biology.

  10. Crystal Structure of the Acyltransferase Domain of the Iterative Polyketide Synthase in Enediyne Biosynthesis*

    PubMed Central

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

    2012-01-01

    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 ATDYN10) 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 Ser651 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

  11. A Single Amino Acid Change Is Responsible for Evolution of Acyltransferase Specificity in Bacterial Methionine Biosynthesis

    SciTech Connect

    Zubieta, C.; Arkus, K.A.J.; Cahoon, R.E.; Jez, J.M.

    2009-05-28

    Bacteria and yeast rely on either homoserine transsuccinylase (HTS, metA) or homoserine transacetylase (HTA; met2) for the biosynthesis of methionine. Although HTS and HTA catalyze similar chemical reactions, these proteins are typically unrelated in both sequence and three-dimensional structure. Here we present the 2.0 {angstrom} resolution x-ray crystal structure of the Bacillus cereus metA protein in complex with homoserine, which provides the first view of a ligand bound to either HTA or HTS. Surprisingly, functional analysis of the B. cereus metA protein shows that it does not use succinyl-CoA as a substrate. Instead, the protein catalyzes the transacetylation of homoserine using acetyl-CoA. Therefore, the B. cereus metA protein functions as an HTA despite greater than 50% sequence identity with bona fide HTS proteins. This result emphasizes the need for functional confirmation of annotations of enzyme function based on either sequence or structural comparisons. Kinetic analysis of site-directed mutants reveals that the B. cereus metA protein and the E. coli HTS share a common catalytic mechanism. Structural and functional examination of the B. cereus metA protein reveals that a single amino acid in the active site determines acetyl-CoA (Glu-111) versus succinyl-CoA (Gly-111) specificity in the metA-like of acyltransferases. Switching of this residue provides a mechanism for evolving substrate specificity in bacterial methionine biosynthesis. Within this enzyme family, HTS and HTA activity likely arises from divergent evolution in a common structural scaffold with conserved catalytic machinery and homoserine binding sites.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol.

    PubMed

    Essemine, Jemâa; Govindachary, Sridharan; Ammar, Saïda; Bouzid, Sadok; Carpentier, Robert

    2011-09-01

    Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5min) mild (40°C) or strong (44°C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.

  19. Oxalic acid and diacylglycerol 36:3 are cross-species markers of sleep debt.

    PubMed

    Weljie, Aalim M; Meerlo, Peter; Goel, Namni; Sengupta, Arjun; Kayser, Matthew S; Abel, Ted; Birnbaum, Morris J; Dinges, David F; Sehgal, Amita

    2015-02-24

    Sleep is an essential biological process that is thought to have a critical role in metabolic regulation. In humans, reduced sleep duration has been associated with risk for metabolic disorders, including weight gain, diabetes, obesity, and cardiovascular disease. However, our understanding of the molecular mechanisms underlying effects of sleep loss is only in its nascent stages. In this study we used rat and human models to simulate modern-day conditions of restricted sleep and addressed cross-species consequences via comprehensive metabolite profiling. Serum from sleep-restricted rats was analyzed using polar and nonpolar methods in two independent datasets (n = 10 per study, 3,380 measured features, 407 identified). A total of 38 features were changed across independent experiments, with the majority classified as lipids (18 from 28 identified). In a parallel human study, 92 metabolites were identified as potentially significant, with the majority also classified as lipids (32 of 37 identified). Intriguingly, two metabolites, oxalic acid and diacylglycerol 36:3, were robustly and quantitatively reduced in both species following sleep restriction, and recovered to near baseline levels after sleep restriction (P < 0.05, false-discovery rate < 0.2). Elevated phospholipids were also noted after sleep restriction in both species, as well as metabolites associated with an oxidizing environment. In addition, polar metabolites reflective of neurotransmitters, vitamin B3, and gut metabolism were elevated in sleep-restricted humans. These results are consistent with induction of peroxisome proliferator-activated receptors and disruptions of the circadian clock. The findings provide a potential link between known pathologies of reduced sleep duration and metabolic dysfunction, and potential biomarkers for sleep loss.

  20. Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase Cα

    PubMed Central

    Stewart, Mikaela D.; Morgan, Brittany; Massi, Francesca; Igumenova, Tatyana I.

    2012-01-01

    C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG-binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of the signaling response and the selectivity of this response among the DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG-binding affinities. In this work, we characterized the C1B domain of Protein Kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or sub-nanosecond dynamics of the protein backbone, but resulted in a >100-fold increase of DAG binding affinity and substantial change in μs-timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between the wild-type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue, Gln128, in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan sidechain into the water-lipid interface are important factors that modulate the DAG-binding properties of C1 domains. PMID:21419781

  1. PTH (parathyroid hormone) elevates inositol polyphosphates and diacylglycerol in a rat osteoblast-like cell line

    SciTech Connect

    Civitelli, R.; Reid, I.R.; Westbrook, S.; Avioli, L.V.; Hruska, K.A. )

    1988-11-01

    Parathyroid hormone (PTH)-stimulated signal transduction through mechanisms alternate to adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) production were studied in UMR 106-01 cells, a cell line with an osteoblastic phenotype. PTH produced transient, dose-related increases in cytosolic calcium ((Ca{sup 2+}){sub i}), inositol trisphosphates, and diacylglycerol (DAG). Both inositol 1,4,5-trisphosphate (Ins-1,4,5P{sub 3}) and inositol 1,3,4-trisphosphate (Ins-1,3,4P{sub 3}) production were rapidly stimulated by PTH. Consistent with the production of Ins-1,3,4P{sub 3}, rapid stimulation of late eluting inositol tetrakisphosphate was observed. The effects on the inositol phosphates were induced rapidly, consistent with roles as signals for changes in (Ca{sup 2+}){sub i}. In saponin-permeabilized UMR 106-01 cells, Ins-1,4,5P{sub 3} stimulated {sup 45}Ca release from a nonmitochondrial intracellular pool. Thus the hypothesis that PTH-stimulated Ins-1,4,5P{sub 3} production initiates Ca{sup 2+} release and contributes to transient elevations of (Ca{sup 2+}){sub i} is supported. These data suggest that stimulation of cAMP production during PTH stimulation may negatively affect production of rises in (Ca{sup 2+}){sub i} during PTH stimulation. The inactivation of the inhibitory G protein of adenylate cyclase by pertussis toxin could explain its action similar to cAMP analogues. Cyclci nucleotides diminish the effects of PTH on (Ca{sup 2+}){sub i}, probably interacting on a biochemical step subsequent to or independent of Ins-1,4,5P{sub 3} release.

  2. Role of diacylglycerol in adrenergic-stimulated sup 86 Rb uptake by proximal tubules

    SciTech Connect

    Baines, A.D.; Drangova, R.; Ho, P. )

    1990-05-01

    We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.

  3. Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

    SciTech Connect

    Griendling, K.K.; Rittenhouse, S.E.; Brock, T.A.; Ekstein, L.S.; Gimbrone, M.A. Jr.; Alexander, R.W.

    1986-05-05

    Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.

  4. Distinct 1-monoacylglycerol and 2-monoacylglycerol kinase activities of diacylglycerol kinase isozymes.

    PubMed

    Sato, Yuriko; Murakami, Chiaki; Yamaki, Atsumi; Mizuno, Satoru; Sakai, Hiromichi; Sakane, Fumio

    2016-09-01

    Diacylglycerol kinase (DGK) consists of ten isozymes and is involved in a wide variety of patho-physiological events. However, the enzymological properties of DGKs have not been fully understood. In this study, we performed a comprehensive analysis on the 1-monoacylglycerol kinase (MGK) and 2-MGK activities of ten DGK isozymes. We revealed that type I (α, β and γ), type II (δ, η and κ) and type III (ε) DGKs have 7.9-19.2% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were <3.0%. Both the 1-MGK and 2-MGK activities of the type IV DGKs (ζ and ι) were <1% relative to their DGK activities. Intriguingly, type V DGKθ has approximately 6% 1-MGK activity and <2% 2-MGK activity compared to its DGK activity. Purified DGKθ exhibited the same results, indicating that its 1-MGK activity is intrinsic. Therefore, DGK isozymes are categorized into three types with respect to their 1-MGK and 2-MGK activities: those having (1) 2-MGK activity relatively stronger than their 1-MGK activity (types I-III), (2) only negligible 1-MGK and 2-MGK activities (type IV), and (3) 1-MGK activity stronger than its 2-MGK activity (type V). The 1-MGK activity of DGKθ and the 2-MGK activity of DGKα were stronger than those of the acylglycerol kinase reported as 1-MGK and 2-MGK to date. The presence or absence of 1-MGK and 2-MGK activities may be essential to the patho-physiological functions of each DGK isozyme.

  5. Substrate selectivity of diacylglycerol kinase in PDGF-stimulated 3T3 cells

    SciTech Connect

    MacDonald, M.L.; Mack, K.F.; Glomset, J.A.

    1987-05-01

    The authors investigated the properties of Diacylglycerol (DAG) Kinase in 3T3 cells. PDGF treatment caused an increase in DAG mass, an increase in incorporation of /sup 32/P into phosphatidic acid (PA) and phosphatidylinositol (PI), and an increase in the rate of phosphorylation of membrane DAG in vitro. The mechanism of enhanced phosphorylation of DAG was studied with dicaprylin (diC/sub 10/) as a probe. Cells were prelabeled with /sup 32/P and treated with PDGF or carrier. DiC/sub 10/ was added to the cell medium before harvesting. With PDGF treatment, the radioactivity in endogenous PA increased fourfold, whereas the radioactivity in PA/sub 10/ and PI/sub 10/ was consistently decreased. To verify that the PDGF effect on PA/sub 10/ formation in intact cells was due to reduced phosphorylation of diC/sub 10/ by DAG kinase, cells were treated with PDGF and/or diC/sub 10/, freeze-thawed, and then incubated with Mg(/sup 32/P)ATP. The rate of phosphorylation of cell-associated diC/sub 10/ was decreased 50% by PDGF treatment. This effect could not be explained by decreased intracellular levels of diC/sub 10/, or by saturation of DAG kinase with endogenous DAGs. Therefore, it seemed that endogenous DAGs, derived from PI, might be better substrates for DAG kinase than is diC/sub 10/. In studies of the properties of DAG kinase with pure DAGs in mixed detergent micelles, they found that the enzyme phosphorylated arachidonoyl-DAG more readily than diC/sub 10/. The selectivity of DAG kinase may play a key role in the formation of arachidonoyl species of PI.

  6. A unique mono- and diacylglycerol lipase from Penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity.

    PubMed

    Tan, Zhong-Biao; Li, Jian-Fang; Li, Xue-Ting; Gu, Ying; Wu, Min-Chen; Wu, Jing; Wang, Jun-Qing

    2014-01-01

    A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5-9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries. PMID:25051359

  7. Protein Kinase Cα (PKCα) Is Resistant to Long Term Desensitization/Down-regulation by Prolonged Diacylglycerol Stimulation.

    PubMed

    Lum, Michelle A; Barger, Carter J; Hsu, Alice H; Leontieva, Olga V; Black, Adrian R; Black, Jennifer D

    2016-03-18

    Sustained activation of PKCα is required for long term physiological responses, such as growth arrest and differentiation. However, studies with pharmacological agonists (e.g. phorbol 12-myristate 13-acetate (PMA)) indicate that prolonged stimulation leads to PKCα desensitization via dephosphorylation and/or degradation. The current study analyzed effects of chronic stimulation with the physiological agonist diacylglycerol. Repeated addition of 1,2-dioctanoyl-sn-glycerol (DiC8) resulted in sustained plasma membrane association of PKCα in a pattern comparable with that induced by PMA. However, although PMA potently down-regulated PKCα, prolonged activation by DiC8 failed to engage known desensitization mechanisms, with the enzyme remaining membrane-associated and able to support sustained downstream signaling. DiC8-activated PKCα did not undergo dephosphorylation, ubiquitination, or internalization, early events in PKCα desensitization. Although DiC8 efficiently down-regulated novel PKCs PKCδ and PKCϵ, differences in Ca(2+) sensitivity and diacylglycerol affinity were excluded as mediators of the selective resistance of PKCα. Roles for Hsp/Hsc70 and Hsp90 were also excluded. PMA, but not DiC8, targeted PKCα to detergent-resistant membranes, and disruption of these domains with cholesterol-binding agents demonstrated a role for differential membrane compartmentalization in selective agonist-induced degradation. Chronic DiC8 treatment failed to desensitize PKCα in several cell types and did not affect PKCβI; thus, conventional PKCs appear generally insensitive to desensitization by sustained diacylglycerol stimulation. Consistent with this conclusion, prolonged (several-day) membrane association/activation of PKCα is seen in self-renewing epithelium of the intestine, cervix, and skin. PKCα deficiency affects gene expression, differentiation, and tumorigenesis in these tissues, highlighting the importance of mechanisms that protect PKCα from

  8. 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

  9. Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Browse, J.; Somerville, C. )

    1988-06-01

    The leaf membrane lipids of many plant species, including Arabidopsis thaliana (L.) Heynh., are synthesized by two complementary pathways that are associated with the chloroplast and the endoplasmic reticulum. By screening directly for alterations in lipid acyl-group composition, the authors have identified several mutants of Arabidopsis that lack the plastid pathway because of a deficiency in activity of the first enzyme in the plastid pathway of glycerolipid synthesis, acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The lesion results in an increased synthesis of lipids by the cytoplasmic pathway that largely compensates for the loss of the plastid pathway and provides nearly normal amounts of all the lipids required for chloroplast biogenesis. However, the fatty acid composition of the leaf membrane lipids of the mutants is altered because the acyltransferases associated with the two pathways normally exhibit different substrate specificities. The remarkable flexibility of the system provides an insight into the nature of the regulatory mechanisms that allocate lipids for membrane biogenesis.

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

    PubMed Central

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

    2016-01-01

    Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and “reset” mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases’ structure–mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins. PMID:27681620

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Conformation and topology of diacylglycerol kinase in E.coli membranes revealed by solid-state NMR spectroscopy.

    PubMed

    Chen, Yanke; Zhang, Zhengfeng; Tang, Xinqi; Li, Jianping; Glaubitz, Clemens; Yang, Jun

    2014-05-26

    Solid-state NMR is a powerful tool for studying membrane proteins in a native-like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid-state NMR are different from those determined by solution-state NMR and X-ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins.

  19. No-carrier-added carbon-11-labeled sn-1,2- and sn-1,3-diacylglycerols by (11C)propyl ketene method

    SciTech Connect

    Imahori, Y.; Fujii, R.; Ueda, S.; Ido, T.; Nishino, H.; Moriyama, Y.; Yamamoto, Y.L.; Nakahashi, H. )

    1991-08-01

    This article describes the preparation of sn-1,2-(11C)diacylglycerols and sn-1,3-(11C)diacylglycerols by a no-carrier-added reaction based on a labeling method using (1-11C)propyl ketene, which is one of the most potent acylating agents. (1-11C)Propyl ketene was produced by pyrolytic decomposition of (1-11C)butyric acid and was trapped in pyridine containing L-alpha-palmitoyl-lysophosphatidylcholine, producing L-alpha-palmitoyl-2-(1-11C)butyryl-sn-glycero-3-phosphorylcholine. The authors adopted an enzymatic reaction to remove the phosphorylcholine, in which L-alpha-palmitoyl-2-(1-11C)butyryl-sn-glycero-3-phosphorylcholine was incubated with phospholipase C, hydrolyzing to produce 1-palmitoyl-sn-2-(1-11C)butyrylglycerol. Total synthesis time was about 50 minutes and the specific activity was estimated at 93 GBq/mumol (2.5 Ci/mumol) at end of synthesis. Radiochemical yield was 3.8% based on the trapped 11CO2. sn-1,3-(11C)Diacylglycerol was also synthesized by (1-11C)propyl ketene reaction with 1-palmitoyl-sn-glycerol in a single procedure. The regional brain tissue radioactivities obtained in sn-1,2-(11C)diacylglycerol were higher than those of sn-1,3-(11C)diacylglycerol, and the regional values varied widely. In autoradiography of brain slices from conscious rats, sn-1,2-(11C)diacylglycerol incorporation sites were discretely localized, especially in the amygdala, cerebral cortex, and hippocampus, suggesting that intensive neuronal processing occurred in these areas on the basis of phosphatidylinositol turnover.

  20. Loss of β-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

    PubMed

    Dixon, Joseph L; Kim, Youn-Kyung; Brinker, Anita; Quadro, Loredana

    2014-01-01

    We provide novel insights into the function(s) of β-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving β-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.

  1. Loss of β-carotene 15,15'-oxygenase in developing mouse tissues alters esterification of retinol, cholesterol and diacylglycerols.

    PubMed

    Dixon, Joseph L; Kim, Youn-Kyung; Brinker, Anita; Quadro, Loredana

    2014-01-01

    We provide novel insights into the function(s) of β-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving β-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue. PMID:23988655

  2. 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

  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. Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes.

    PubMed

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

    2009-12-01

    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 synthetic lethal 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.

  5. Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases

    PubMed Central

    Klein, Gracjana; Lindner, Buko; Brabetz, Werner; Brade, Helmut; Raina, Satish

    2009-01-01

    To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Δ(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 °C, besides showing accumulation of free lipid IVA precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Δ(waaC lpxL lpxM lpxP) strains showed primarily Kdo2-lipid IVA, indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 °C. These lipid IVA derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. ΔwaaA and Δ(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and σE signal transduction pathways. Δ(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Δ(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Δ(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II. PMID:19346244

  6. Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements

    SciTech Connect

    Koynova, Rumiana; Tihova, Mariana

    2010-04-12

    Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20-30 wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.

  7. Novel Conjugates of 1,3-Diacylglycerol and Lipoic Acid: Synthesis, DPPH Assay, and RP-LC-MS-APCI Analysis

    PubMed Central

    Madawala, Samanthi R. P.; Andersson, Rolf E.; Jastrebova, Jelena A.; Almeida, Maria; Dutta, Paresh C.

    2011-01-01

    1,3-Diacylglycerol is known to reduce body weight and fat deposits in humans. α-Lipoic acid is a potent antioxidant and effective against many pathological conditions, including obesity and related metabolic syndromes. The present work is based on the hypothesis that the hybrid molecules of 1,3-diacylglycerol and lipoic acid possess synergistic and/or additive effects compared with the parent compounds against obesity, overweight, and related metabolic syndromes. Laboratory scale synthesis of 1,3-dioleoyl-2-lipoyl-sn-glycerol (yield 80%) and 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (yield 70%) was performed for the first time and supported by NMR and MS data. Free radical scavenging capacity of the conjugates was assayed using DPPH test. A remarkably high in vitro free radical scavenging capacity was demonstrated for the 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (EC50 value 0.21). RP-HPLC-MS-APCI analysis showed satisfactory separation between the conjugates (R~1). Protonated molecular ion of the conjugates at m/z 809 and m/z at 811, respectively, and their characteristic fragment ions were abundant. PMID:21966595

  8. Diacylglycerol-Rich Domain Formation in Giant Stearoyl-Oleoyl Phosphatidylcholine Vesicles Driven by Phospholipase C Activity

    PubMed Central

    Riske, Karin A.; Döbereiner, Hans-Günther

    2003-01-01

    We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (α-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs. PMID:14507699

  9. Diacylglycerol kinase θ couples farnesoid X receptor-dependent bile acid signalling to Akt activation and glucose homoeostasis in hepatocytes.

    PubMed

    Cai, Kai; Sewer, Marion B

    2013-09-01

    DGKs (diacylglycerol kinases) catalyse the conversion of diacylglycerol into PA (phosphatidic acid), a positive modulator of mTOR (mammalian target of rapamycin). We have found that chenodeoxycholic acid and the synthetic FXR (farnesoid X receptor) ligand GW4064 induce the mRNA and protein expression of DGKθ in the HepG2 cell line and in primary human hepatocytes. Reporter gene studies using 1.5 kB of the DGKθ promoter fused to the luciferase gene revealed that bile acids increase DGKθ transcriptional activity. Mutation of putative FXR-binding sites attenuated the ability of GW4046 to increase DGKθ luciferase activity. Consistent with this finding, ChIP (chromatin immunoprecipitation) assays demonstrated that bile acid signalling increased the recruitment of FXR to the DGKθ promoter. Furthermore, GW4064 evoked a time-dependent increase in the cellular concentration of PA. We also found that GW4064 and PA promote the phosphorylation of mTOR, Akt and FoxO1 (forkhead box O1), and that silencing DGKθ expression significantly abrogated the ability of GW4046 to promote the phosphorylation of these PA-regulated targets. DGKθ was also required for bile-acid-dependent decreased glucose production. Taken together, our results establish DGKθ as a key mediator of bile-acid-stimulated modulation of mTORC2 (mTOR complex 2), the Akt pathway and glucose homoeostasis.

  10. Diacylglycerol kinase-ζ regulates mTORC1 and lipogenic metabolism in cancer cells through SREBP-1

    PubMed Central

    Torres-Ayuso, P; Tello-Lafoz, M; Mérida, I; Ávila-Flores, A

    2015-01-01

    Diacylglycerol kinases (DGKs) transform diacylglycerol (DAG) into phosphatidic acid (PA), balancing the levels of these key metabolic and signaling lipids. We previously showed that PA derived from the DGKζ isoform promotes mammalian target of rapamycin complex 1 (mTORC1) activation. This function might be crucial for the growth and survival of cancer cells, especially for those resistant to the allosteric mTOR inhibitor rapamycin. How this positive function of DGKζ coordinates with DAG metabolism and signaling is unknown. In this study, we used a rapamycin-resistant colon cancer cell line as a model to address the role of DGKζ in tumor cells. We found that DGKζ predominated over other PA sources such as DGKα or phospholipase D to activate mTORC1, and that its activity was a component of the rapamycin-induced feedback loops. We show that the DGKζ DAG-consuming function is central to cell homeostasis, as DAG negatively regulates levels of the lipogenic transcription factor SREBP-1. Our findings suggest a model in which simultaneous regulation of DAG and PA levels by DGKζ is integrated with mTOR function to maintain tumor cell homeostasis; we provide new evidence of the crosstalk between mTOR and lipid metabolism that will be advantageous in the design of drug therapies. PMID:26302180

  11. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-02-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

  12. Redundant and specialized roles for diacylglycerol kinases α and ζ in the control of T cell functions.

    PubMed

    Mérida, Isabel; Andrada, Elena; Gharbi, Severine I; Ávila-Flores, Antonia

    2015-04-28

    The diacylglycerol kinases (DGKs) attenuate diacylglycerol (DAG)-mediated signals by catalyzing the conversion of DAG to phosphatidic acid. In T lymphocytes, the antigen-stimulated generation of DAG links signal strength to the intensity and duration of signaling by the Ras-extracellular signal-regulated kinase (ERK) and protein kinase C (PKC)-dependent pathways. The generation of DAG at the plasma membrane of T cells lies at the core of the mechanisms that delimit T cell functions. DGKα and DGKζ are the two main isoforms that are found in T cells, and several approaches define their precise contribution to T cell responses. Each of these isoforms has specialized and redundant functions that limit the intensity of DAG-regulated signals downstream of antigenic stimulation. This ability, which in normal T cells contributes to maintaining homeostasis and function, is exploited by tumors to evade immune surveillance. Modification of DGK activity offers new perspectives for the therapeutic manipulation of T cell functions for treatment of autoimmune pathologies, or for overcoming tumor-induced T cell tolerance. Precise knowledge of the mechanisms that sustain DGK isoform-specific regulation in T lymphocytes is indispensable for the development of new tools for pharmacological intervention.

  13. Involvement of diacylglycerol kinase β in the spine formation at distal dendrites of striatal medium spiny neurons.

    PubMed

    Hozumi, Yasukazu; Kakefuda, Kenichi; Yamasaki, Miwako; Watanabe, Masahiko; Hara, Hideaki; Goto, Kaoru

    2015-01-12

    Spine formation, a salient feature underlying neuronal plasticity to adapt to a changing environment, is regulated by complex machinery involving membrane signal transduction. The diacylglycerol kinase (DGK) family, which is involved in membrane lipid metabolism, catalyzes the phosphorylation of a lipid second messenger, diacylglycerol (DG). Of the DGKs, DGKβ is characterized by predominant expression in a specific brain region: the striatum. We previously demonstrated that DGKβ is expressed selectively in medium spiny neurons (MSNs) and that it is highly enriched in the perisynaptic membrane on dendritic spines contacted with excitatory terminals. Moreover, DGKβ regulates spinogenesis through actin-based remodeling in an activity-dependent manner. However, the detailed mechanisms of spinogenesis regulation and its functional significance remain unclear. To address these issues, we performed Golgi-Cox staining to examine morphological aspects of MSNs in the striatum of DGKβ-knockout (KO) mice. Results show that striatal MSNs of DGKβ-KO mice exhibited lower dendritic spine density at distal dendrites than wild-type mice did. We also sought protein targets that interact with DGKβ and identified the GluA2 AMPA receptor subunit as a novel DGKβ binding partner. In addition, DGKβ-deficient brain exhibits significant reduction of TARP γ-8, which represents a transmembrane AMPA receptor regulatory protein. These findings suggest that DGKβ regulates the spine formation at distal dendrites in MSNs.

  14. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src.

    PubMed

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-10-30

    Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

  15. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src

    PubMed Central

    Torres-Ayuso, Pedro; Daza-Martín, Manuel; Martín-Pérez, Jorge; Ávila-Flores, Antonia; Mérida, Isabel

    2014-01-01

    Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer. PMID:25339152

  16. Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism.

    PubMed Central

    Gordon, S. E.; Downing-Park, J.; Tam, B.; Zimmerman, A. L.

    1995-01-01

    The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically interferes with channel opening, and raise the question of whether DAG is involved in visual transduction. PMID:8527654

  17. Diacylglycerol, phosphatidic acid, and their metabolic enzymes in synaptic vesicle recycling.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Raben, Daniel M

    2015-01-01

    The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling of vesicles, and subsequent release of the refilled vesicles from the presynaptic bouton. SV exocytosis is tightly linked with endocytosis, and variations in the number of vesicles, and/or defects in the refilling of SVs, will affect the amount of neurotransmitter available for release (Sudhof, 2004). There is increasing interest in the roles synaptic vesicle lipids and lipid metabolizing enzymes play in this recycling. Initial emphasis was placed on the role of polyphosphoinositides in SV cycling as outlined in a number of reviews (Lim and Wenk, 2009; Martin, 2012; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Other lipids are now recognized to also play critical roles. For example, PLD1 (Humeau et al., 2001; Rohrbough and Broadie, 2005) and some DGKs (Miller et al., 1999; Nurrish et al., 1999) play roles in neurotransmission which is consistent with the critical roles for phosphatidic acid (PtdOH) and diacylglycerol (DAG) in the regulation of SV exo/endocytosis (Cremona et al., 1999; Exton, 1994; Huttner and Schmidt, 2000; Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). PLD generates phosphatidic acid by catalyzing the hydrolysis of phosphatidylcholine (PtdCho) and in some systems this PtdOH is de-phosphorylated to generate DAG. In contrast, DGK catalyzes the phosphorylation of DAG thereby converting it into PtdOH. While both enzymes are poised to regulate the levels of DAG and PtdOH, therefore, they both lead to the generation of PtdOH and could have opposite effects on DAG levels. This is particularly important for SV cycling as PtdOH and DAG are both needed for evoked exocytosis (Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Two lipids and their involved metabolic enzymes, two sphingolipids have also been implicated in

  18. Diacylglycerol kinase epsilon in bovine and rat photoreceptor cells. Light-dependent distribution in photoreceptor cells.

    PubMed

    Natalini, Paola M; Zulian, Sandra E; Ilincheta de Boschero, Mónica G; Giusto, Norma M

    2013-07-01

    The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKɛ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKɛ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKɛ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [(3)H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-(32)P]ATP and 1-stearoyl, 2-arachidonoylglycerol (SAG), was found to be significantly higher in BBROS than in BDROS. Higher light-dependent DAGK activity (condition b) was also found when ROS were isolated from dark-adapted rat retinas exposed to light. Western blot analysis of isolated ROS proteins from bovine and rat retinas confirmed that illumination increases DAGKɛ content in the outer segments of these two species. Light-dependent DAGKɛ localization in the outer segment was not observed when U73122, a phospholipase C inhibitor, was present prior to the exposure of rat eyecups (in situ model) to light. Furthermore, no increased PA synthesis from [(3)H]DAG and ATP was observed in the presence of neomycin prior to the exposure of bovine eyecups to light. Interestingly, when BBROS were pre-phosphorylated with ATP in the presence of 1,2-dioctanoyl sn-glycerol (di-C8) or phorbol dibutyrate (PDBu) as PKC activation conditions, higher DAGK activity was observed than in dephosphorylated controls. Taken together, our findings suggest that the selective distribution of DAGKɛ in photoreceptor cells is a light-dependent mechanism that promotes increased SAG removal and synthesis of 1-stearoyl, 2-arachidonoyl phosphatidic acid in the sensorial portion of this cell, thus demonstrating a novel mechanism of light

  19. Postsynaptic diacylglycerol lipase α mediates retrograde endocannabinoid suppression of inhibition in mouse prefrontal cortex

    PubMed Central

    Yoshino, Hiroki; Miyamae, Takeaki; Hansen, Gwenn; Zambrowicz, Brian; Flynn, Michael; Pedicord, Donna; Blat, Yuval; Westphal, Ryan S; Zaczek, Robert; Lewis, David A; Gonzalez-Burgos, Guillermo

    2011-01-01

    Abstract Depolarization-induced suppression of inhibition (DSI) is a prevailing form of endocannabinoid signalling. However, several discrepancies have arisen regarding the roles played by the two major brain endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide, in mediating DSI. Here we studied endocannabinoid signalling in the prefrontal cortex (PFC), where several components of the endocannabinoid system have been identified, but endocannabinoid signalling remains largely unexplored. In voltage clamp recordings from mouse PFC pyramidal neurons, depolarizing steps significantly suppressed IPSCs induced by application of the cholinergic agonist carbachol. DSI in PFC neurons was abolished by extra- or intracellular application of tetrahydrolipstatin (THL), an inhibitor of the 2-AG synthesis enzyme diacylglycerol lipase (DAGL). Moreover, DSI was enhanced by inhibiting 2-AG degradation, but was unaffected by inhibiting anandamide degradation. THL, however, may affect other enzymes of lipid metabolism and does not selectively target the α (DAGLα) or β (DAGLβ) isoforms of DAGL. Therefore, we studied DSI in the PFC of DAGLα−/− and DAGLβ−/− mice generated via insertional mutagenesis by gene-trapping with retroviral vectors. Gene trapping strongly reduced DAGLα or DAGLβ mRNA levels in a locus-specific manner. In DAGLα−/− mice cortical levels of 2-AG were significantly decreased and DSI was completely abolished, whereas DAGLβ deficiency did not alter cortical 2-AG levels or DSI. Importantly, cortical levels of anandamide were not significantly affected in DAGLα−/− or DAGLβ−/− mice. The chronic decrease of 2-AG levels in DAGLα−/− mice did not globally alter inhibitory transmission or the response of cannabinoid-sensitive synapses to cannabinoid receptor stimulation, although it altered some intrinsic membrane properties. Finally, we found that repetitive action potential firing of PFC pyramidal neurons suppressed synaptic

  20. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid

    SciTech Connect

    Takuwa, Y.; Takuwa, N.; Rasmussen, H.

    1986-11-05

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of (/sup 3/H)inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in (/sup 3/H)inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of (/sup 3/H)inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca/sup 2 +/ and does not appear to be secondary to an increase in intracellular Ca/sup 2 +/. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  1. Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid.

    PubMed

    Takuwa, Y; Takuwa, N; Rasmussen, H

    1986-11-01

    The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2-diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C-mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2-diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2-diacylglycerol.

  2. Optimum conditions for the production of soy polyol oils and diacylglycerol from soybean oil by Acinetobacter haemolyticus A01-35 NRRL B-59985

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Triacylglycerols (TAG) containing hydroxy fatty acids have many industrial uses such as the manufacture of aviation lubricant, plastic, paint, nylons, and cosmetics, because of the hydroxyl groups on the fatty acid (FA) constituents. Diacylglycerols (DAG) containing hydroxy FA can also be used in th...

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

    PubMed

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

    2016-07-12

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

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

    PubMed

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

    2002-03-13

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

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

    PubMed Central

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

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

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

    PubMed

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

    2000-12-01

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

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

    PubMed

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

    2001-03-01

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

  8. TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2[S

    PubMed Central

    Pramfalk, Camilla; Melhuish, Tiffany A.; Wotton, David; Jiang, Zhao-Yan; Eriksson, Mats; Parini, Paolo

    2014-01-01

    Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism. PMID:24478032

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

    PubMed

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

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

  10. Lecithin cholesterol acyltransferase (LCAT) activity as a predictor for ketosis and parturient haemoglobinuria in Egyptian water buffaloes.

    PubMed

    Ghanem, Mohamed M; El-Deeb, Wael M

    2010-02-01

    Lecithin cholesterol acyltransferase (LCAT) activity was measured in 48 Egyptian water buffaloes four weeks pre-parturient. The activity was significantly low in 37 buffaloes (77.1%). Four weeks post-partum, clinical examination revealed that 23 buffaloes had the clinical signs of ketosis (K) while 14 had the clinical signs of parturient-haemoglobinuria (PHU). Serum samples were collected from 5 buffaloes of each group (K and PHU) besides 5 clinically healthy buffaloes with normal LCAT (control). Glucose level was significantly reduced in K and PHU groups while the phosphorous (P) level was significantly reduced in PHU group compared to control. There were significant reductions in the total cholesterol, free cholesterol, triglycerides, total protein and albumin in K and PHU groups; whereas, significant increases in AST, GGT, non-esterified fatty acids (NEFA) and beta-hydroxybutyric acid (BHBA) in K and PHU groups were detected. Therefore, LCAT could be a predictor for metabolic disorders in Egyptian water buffaloes.

  11. Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.

    PubMed Central

    Brown, Adrian P; Carnaby, Simon; Brough, Clare; Brazier, Melissa; Slabas, Antoni R

    2002-01-01

    Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and

  12. Essential Role of Lysophosphatidylcholine Acyltransferase 3 in the Induction of Macrophage Polarization in PMA-Treated U937 Cells.

    PubMed

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

    2015-12-01

    Lysophospholipid acyltransferases (LPLATs) regulate the diversification of fatty acid composition in biological membranes. Lysophosphatidylcholine acyltransferases (LPCATs) are members of the LPLATs that play a role in inflammatory responses. M1 macrophages differentiate in response to lipopolysaccharide (LPS) and are pro-inflammatory, whereas M2 macrophages, which differentiate in response to interleukin-4 (IL-4), are anti-inflammatory and involved in homeostasis and wound healing. In the present study, we showed that LPCATs play an important role in M1/M2-macrophage polarization. LPS changed the shape of PMA-treated U937 cells from rounded to spindle shaped and upregulated the mRNA and protein expression of the M1 macrophage markers CXCL10, TNF-α, and IL-1β. IL-4 had no effect on the shape of PMA-treated U937 cells and upregulated the M2 macrophage markers CD206, IL-1ra, and TGF-β in PMA-treated U937 cells. These results suggest that LPS and IL-4 promote the differentiation of PMA-treated U937 cells into M1- and M2-polarized macrophages, respectively. LPS significantly downregulated the mRNA expression of LPCAT3, one of four LPCAT isoforms, and suppressed its enzymatic activity toward linoleoyl-CoA and arachidonoyl-CoA in PMA-treated U937 cells. LPCAT3 knockdown induced a spindle-shaped morphology typical of M1-polarized macrophages, and increased the secretion of CXCL10 and decreased the levels of CD206 in IL-4-activated U937 cells. This indicates that knockdown of LPCAT3 shifts the differentiation of PMA-treated U937 cells to M1-polarized macrophages. Our findings suggest that LPCAT3 plays an important role in M1/M2-macrophage polarization, providing novel potential therapeutic targets for the regulation of immune and inflammatory disorders.

  13. Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

    PubMed

    Oancea, E; Teruel, M N; Quest, A F; Meyer, T

    1998-02-01

    Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

  14. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells.

    PubMed

    Peddie, Christopher J; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O'Toole, Peter; Larijani, Banafshe; Collinson, Lucy M

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure.

  15. Inhibition of the effects of thrombin on guinea pig platelets by the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.; Sutherland, C.A.; Khandwala, A.S.; Jamall, I.S.; Kapoor, A.L.

    1986-10-01

    Phospholipase C (PLC) and diacylglycerol lipase (DGL) activities were found in guinea pig platelet microsome preparations. No phospholipase A2 (PLA2) activity was detected. RHC 80267 (1,6-di (0-(carbamoyl) cyclohexanone oxime)hexane) inhibited DGL activity (IC50 = 4 uM) from guinea pig platelet microsomes but had no effect on PLC. RHC 80267 inhibited platelet aggregation (IC50 = 11 uM), release of arachidonic acid (AA), its metabolites, and ATP (IC50 = 4.5 uM) when guinea pig platelets were challenged with a low concentration of thrombin. We propose that PLC-DGL is an important enzymatic pathway for the release of AA in guinea pig platelets.

  16. A study of the high pressure phase transition of diacylglycerol oil by means of light transmission and scattering

    NASA Astrophysics Data System (ADS)

    Kościesza, R.; Tefelski, D. B.; Ptasznik, S.; Rostocki, A. J.; Malanowski, A.; Siegoczyński, R. M.

    2012-06-01

    A sample of diacylglycerol (DAG) oil, being a mixture of DAGs and triacylglycerols with a ratio of 82% and 18%, respectively, and with a vestigial content of monoacylglycerols and free fatty acids, has been researched in high pressure conditions. As a result of dynamically applied pressure, a discontinuous (first-order) phase transition of the DAG sample has been observed. After the threshold value of the pressure for the phase change was determined, a number of further measurements were performed. The aim of the measurements was to pressurise the DAG sample with different rates of compression. During the measurements, the following parameters were recorded: the intensities of beams both transmitted and scattered at the angle of 90°, and the temperature of the sample. The obtained data may prove useful for an introductory description of the phase change kinetics induced by dynamically applied pressure.

  17. Prolactin-stimulated ornithine decarboxylase induction in rat hepatocytes: Coupling to diacylglycerol generation and protein kinase C

    SciTech Connect

    Buckley, A.R.; Buckley, D.J. )

    1991-01-01

    The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased ({sup 3}H)-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle.

  18. Endothelin stimulates a sustained 1,2-diacylglycerol increase and protein kinase C activation in bovine aortic smooth muscle cells

    SciTech Connect

    Lee, T.S.; Chao, T.; Hu, K.Q.; King, G.L.

    1989-07-14

    Endothelin is a long-lasting potent vasoconstrictor peptide. We report here that in bovine aortic smooth muscle cells, endothelin biphasically increased total cellular diacylglycerol (DAG) content. When cellular DAG was labeled with (/sup 14/C) glycerol for 48h, endothelin stimulated (/sup 14/C)DAG formation in a biphasic pattern. Only one prolonged phase of DAG accumulation was observed when cells were labeled with (/sup 3/H)glycerol for 2 h. Endothelin induced an increase in the membranous protein kinase C (PKC) activities, which lasted for more than 20 min. These data suggest that (i) endothelin stimulates a sustained generation of DAG, (ii) this accumulation of DAG results in a sustained translocation of cytosolic PKC activities to the membrane.

  19. Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion.

    PubMed

    Abramovici, Hanan; Gee, Stephen H

    2007-07-01

    The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion. PMID:17410543

  20. Diacylglycerol Kinase ζ (DGKζ) Is a Critical Regulator of Bone Homeostasis Via Modulation of c-Fos Levels in Osteoclasts.

    PubMed

    Zamani, Ali; Decker, Corinne; Cremasco, Viviana; Hughes, Lindsey; Novack, Deborah V; Faccio, Roberta

    2015-10-01

    Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.

  1. Diacylglycerol kinase ζ (DGKζ) is a critical regulator of bone homeostasis via modulation of c-Fos levels in osteoclasts†

    PubMed Central

    Zamani, Ali; Decker, Corinne; Cremasco, Viviana; Hughes, Lindsey; Novack, Deborah V.; Faccio, Roberta

    2015-01-01

    Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ−/− mice are osteoporotic due to a marked increase in OC numbers. In vitro, DGKζ−/− bone marrow macrophages (BMMs) form more numerous, larger and highly resorptive OCs. Surprisingly, while increased DAG levels do not alter RANK/RANKL osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine M-CSF. We find that M-CSF is responsible for increased DGKζ−/− OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ−/− cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2+/− BMMs, which have reduced DAG levels and form fewer OCs due to impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2+/− mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression. PMID:25891971

  2. The functional size of acyl-coenzyme A (CoA):cholesterol acyltransferase and acyl-CoA hydrolase as determined by radiation inactivation

    SciTech Connect

    Billheimer, J.T.; Cromley, D.A.; Kempner, E.S. )

    1990-05-25

    Frozen rat liver microsomes and rough endoplasmic reticulum were irradiated with high energy electrons. The surviving enzymatic activity of acyl-CoA:cholesterol acyltransferase and activity for esterification of 25-hydroxycholesterol decreased as a simple exponential function of radiation exposure, leading to a target size of 170-180 kDa. The loss of acyl-CoA hydrolase activity with a radiation dose was complex and resolved as a 45-kDa enzyme associated with a large inhibitor. It is interpreted that acyl-CoA hydrolase is the acyl-CoA-binding component and the inhibitor is the cholesterol-binding component of acyl-CoA:cholesterol acyltransferase.

  3. Identification of an Arabidopsis Fatty Alcohol:Caffeoyl-Coenzyme A Acyltransferase Required for the Synthesis of Alkyl Hydroxycinnamates in Root Waxes1[W][OA

    PubMed Central

    Kosma, Dylan K.; Molina, Isabel; Ohlrogge, John B.; Pollard, Mike

    2012-01-01

    While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots. These extracts include esters of saturated long-chain primary alcohols and hydroxycinnamic acids. Such fatty alcohols and hydroxycinnamic acids are also present in suberin. We demonstrate that alkyl coumarates and caffeates, which are the major components of Arabidopsis (Arabidopsis thaliana) root waxes, are present primarily in taproots. Previously we identified ALIPHATIC SUBERIN FERULOYL TRANSFERASE (At5g41040), a HXXXD-type acyltransferase (BAHD family), responsible for incorporation of ferulate into aliphatic suberin of Arabidopsis. However, aliphatic suberin feruloyl transferase mutants were unaffected in alkyl hydroxycinnamate ester root wax composition. Here we identify a closely related gene, At5g63560, responsible for the synthesis of a subset of alkyl hydroxycinnamate esters, the alkyl caffeates. Transgenic plants harboring PAt5g63560::YFP fusions showed transcriptional activity in suberized tissues. Knockout mutants of At5g63560 were severely reduced in their alkyl caffeate but not alkyl coumarate content. Recombinant At5g63560p had greater acyltransferase activity when presented with caffeoyl-Coenzyme A (CoA) substrate, thus we have named this acyltransferase FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE. Stress experiments revealed elevated alkyl coumarate content in root waxes of NaCl-treated wild-type and fatty alcohol:caffeoyl-CoA caffeoyl transferase plants. We further demonstrate that FATTY ACYL-CoA REDUCTASEs (FARs) FAR5 (At3g44550), FAR4 (At3g44540), and FAR1 (At5g22500) are required for the synthesis of C18, C20, and C22 alkyl hydroxycinnamates, respectively. Collectively, these results suggest that multiple acyltransferases are utilized for the synthesis of alkyl hydroxycinnamate esters of Arabidopsis root waxes and that FAR1/4/5 provide the fatty alcohols required for alkyl hydroxycinnamate synthesis. PMID:22797656

  4. Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes.

    PubMed

    Kosma, Dylan K; Molina, Isabel; Ohlrogge, John B; Pollard, Mike

    2012-09-01

    While suberin is an insoluble heteropolymer, a number of soluble lipids can be extracted by rapid chloroform dipping of roots. These extracts include esters of saturated long-chain primary alcohols and hydroxycinnamic acids. Such fatty alcohols and hydroxycinnamic acids are also present in suberin. We demonstrate that alkyl coumarates and caffeates, which are the major components of Arabidopsis (Arabidopsis thaliana) root waxes, are present primarily in taproots. Previously we identified ALIPHATIC SUBERIN FERULOYL TRANSFERASE (At5g41040), a HXXXD-type acyltransferase (BAHD family), responsible for incorporation of ferulate into aliphatic suberin of Arabidopsis. However, aliphatic suberin feruloyl transferase mutants were unaffected in alkyl hydroxycinnamate ester root wax composition. Here we identify a closely related gene, At5g63560, responsible for the synthesis of a subset of alkyl hydroxycinnamate esters, the alkyl caffeates. Transgenic plants harboring P(At5g63560)::YFP fusions showed transcriptional activity in suberized tissues. Knockout mutants of At5g63560 were severely reduced in their alkyl caffeate but not alkyl coumarate content. Recombinant At5g63560p had greater acyltransferase activity when presented with caffeoyl-Coenzyme A (CoA) substrate, thus we have named this acyltransferase FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE. Stress experiments revealed elevated alkyl coumarate content in root waxes of NaCl-treated wild-type and fatty alcohol:caffeoyl-CoA caffeoyl transferase plants. We further demonstrate that FATTY ACYL-CoA REDUCTASEs (FARs) FAR5 (At3g44550), FAR4 (At3g44540), and FAR1 (At5g22500) are required for the synthesis of C18, C20, and C22 alkyl hydroxycinnamates, respectively. Collectively, these results suggest that multiple acyltransferases are utilized for the synthesis of alkyl hydroxycinnamate esters of Arabidopsis root waxes and that FAR1/4/5 provide the fatty alcohols required for alkyl hydroxycinnamate synthesis.

  5. Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. II. Structure elucidation of pyripyropenes A, B, C and D.

    PubMed

    Kim, Y K; Tomoda, H; Nishida, H; Sunazuka, T; Obata, R; Omura, S

    1994-02-01

    The structures of pyripyropenes A, B, C and D, novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors, were determined mainly by spectroscopic studies including various NMR measurements. Pyripyropenes have a common structure which consists of pyridine, alpha-pyrone and sesquiterpene moieties. One of the three O-acetyl residues in the sesquiterpene moiety of pyripyropene A is replaced with an O-propionyl residue in pyripyropenes B, C and D. PMID:8150710

  6. iso-Migrastatin, Migrastatin, and Dorrigocin Production in Streptomyces platensis NRRL 18993 Is Governed by a Single Biosynthetic Machinery Featuring an Acyltransferase-less Type I Polyketide Synthase*

    PubMed Central

    Lim, Si-Kyu; Ju, Jianhua; Zazopoulos, Emmanuel; Jiang, Hui; Seo, Jeong-Woo; Chen, Yihua; Feng, Zhiyang; Rajski, Scott R.; Farnet, Chris M.; Shen, Ben

    2009-01-01

    iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by λ-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ΔmgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments. PMID:19726666

  7. Formation of an ordered phase by ceramides and diacylglycerols in a fluid phosphatidylcholine bilayer--Correlation with structure and hydrogen bonding capacity.

    PubMed

    Ekman, Peik; Maula, Terhi; Yamaguchi, Shou; Yamamoto, Tetsuya; Nyholm, Thomas K M; Katsumura, Shigeo; Slotte, J Peter

    2015-10-01

    Ceramides and diacylglycerols are lipids with a large hydrophobic part (acyl chains and long-chain base) whereas their polar function (hydroxyl group) is small. They need colipids with large head groups to coexist in bilayer membranes. In this study, we have determined how saturated and unsaturated ceramides and acyl-chain matched diacylglycerols form ordered domains in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers as a function of bilayer concentration. The formation of ordered domains was determined from lifetime analysis of trans-parinaric acid. Ceramides formed ordered domains with equal average tPA lifetime at lower bilayer concentration when compared to acyl-chain matched diacylglycerols. This was true for both saturated (16:0) and mono-unsaturated (18:1) species. This finding suggested that hydrogen bonding among ceramides contributed to their more efficient ordered phase formation, since diacylglycerols do not form similar hydrogen bonding networks. The role of hydrogen bonding in ordered domain formation was further verified by using palmitoyl ceramide analogs with 2N and 3OH methylated long-chain bases. These analogs do not form hydrogen bonds from the 2NH or the 3OH, respectively. While methylation of the 3OH did not affect ordered phase formation compared to native palmitoyl ceramide, 2NH methylation markedly attenuated ceramide ordered phase formation. We conclude that in addition to acyl chain length, saturation, molecular order, and lack of large head group, also hydrogen bonding involving the 2NH is crucial for efficient formation of ceramide-rich domains in fluid phosphatidylcholine bilayers.

  8. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport.

    PubMed

    Cleland, P J; Appleby, G J; Rattigan, S; Clark, M G

    1989-10-25

    Contraction-induced translocation of protein kinase C (Richter E.A., Cleland, P.J.F., Rattigan, S., and Clark, M.G. (1987) FEBS Lett. 217, 232-236) implies a role for this enzyme in muscle contraction or the associated metabolic adjustments. In the present study, this role is further examined particularly in relation to changes in glucose transport. Electrical stimulation of the sciatic nerve of the anesthetized rat in vivo led to a time-dependent translocation of protein kinase C and a 2-fold increase in the concentrations of both diacylglycerol and phosphatidic acid. Maximum values for the latter were reached at 2 min and preceded the maximum translocation of protein kinase C (10 min). Stimulation of muscles in vitro increased the rate of glucose transport, but this required 20 min to reach maximum. There was no reversal of translocation or decrease in the concentrations of diacylglycerol and phosphatidic acid even after 30 min of rest following a 5-min period of stimulation in vivo. Translocation was not influenced by variations in applied load at maximal fiber recruitment but was dependent on the frequency of nontetanic stimuli, reaching a maximum at 4 Hz. The relationship between protein kinase C and glucose transport was also explored by varying the number of tetanic stimuli. Whereas only one train of stimuli (200 ms, 100 Hz) was required for maximal effects on protein kinase C, diacylglycerol, and phosphatidic acid, more than 35 trains of stimuli were required to activate glucose transport. It is concluded that the production of diacylglycerol and the translocation of protein kinase C may be causally related. However, if the translocated protein kinase C is involved in the activation of glucose transport during muscle contractions, an accumulated exposure to Ca2+, resulting from multiple contractions, would appear to be necessary.

  9. Chlamydia trachomatis growth depends on eukaryotic cholesterol esterification and is affected by Acyl-CoA:cholesterol acyltransferase inhibition

    PubMed Central

    Peters, Jan; Byrne, Gerald I.

    2015-01-01

    Chlamydia trachomatis is auxotrophic for a variety of essential metabolites. Inhibitors that interrupt host cell catabolism may inhibit chlamydial growth and reveal Chlamydia metabolite requirements. We used the known indoleamine-2,3-dioxygenase (IDO)-inhibitor 4-phenyl imidazole (4-PI) to reverse Interferon (IFN)-γ-induced chlamydial growth inhibition. However, at elevated inhibitor concentrations chlamydial growth was arrested even in the absence of IFN-γ. Since 4-PI is known to interfere with cholesterol metabolism, the effect of cholesterol add-back was tested. Chlamydia growth was restored in the presence of cholesterol in serum-containing, but not serum-free medium suggesting that cholesterol and other serum components are required for growth recovery. When serum factors were tested, either cholesteryl linoleate or the combination of cholesterol and linoleic acid restored chlamydial growth. However, growth was not restored when either cholesterol or linoleic acid were added alone, suggesting that the production of cholesteryl esters from cholesterol and fatty acids was affected by 4-PI treatment. In eukaryotic cells, the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the production of cholesteryl esters. When HeLa cells were treated with the ACAT-specific inhibitor 4-hydroxycinnamicacid amide C. trachomatis growth was interrupted, but was restored by the addition of cholesteryl linoleate, suggesting that ACAT activity is necessary for intracellular Chlamydia growth. PMID:25883118

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

    SciTech Connect

    Atkinson, E.M.; Long, S.R. ); Palcic, M.M.; Hindsgaul, O. )

    1994-08-30

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

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

    PubMed Central

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

    2015-01-01

    Cotton (Gossypium spp.) is an important economic crop and there is obvious heterosis in cotton, fertility has played an important role in this heterosis. However, the genes that exhibit critical roles in anther development and fertility are not well understood. Here, we report an acyl-CoA N-acyltransferase (EC2.3; GhACNAT) that plays a key role in anther development and fertility. Suppression of GhACNAT by virus-induced gene silencing in transgenic cotton (G. hirsutum L. cv. C312) resulted in indehiscent anthers that were full of pollen, diminished filaments and stamens, and plant sterility. We found GhACNAT was involved in lipid metabolism and jasmonic acid (JA) biosynthesis. The genes differentially expressed in GhACNAT-silenced plants and C312 were mainly involved in catalytic activity and transcription regulator activity in lipid metabolism. In GhACNAT-silenced plants, the expression levels of genes involved in lipid metabolism and jasmonic acid biosynthesis were significantly changed, the amount of JA in leaves and reproductive organs was significantly decreased compared with the amounts in C312. Treatments with exogenous methyl jasmonate rescued anther dehiscence and pollen release in GhACNAT-silenced plants and caused self-fertility. The GhACNAT gene may play an important role in controlling cotton fertility by regulating the pathways of lipid synthesis and JA biogenesis. PMID:26134787

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

    PubMed

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

    2010-01-01

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

  13. TRANSCRIPTIONAL REGULATION OF MITOCHONDRIAL GLYCEROPHOSPHATE ACYLTRANSFERASE IS MEDIATED BY DISTAL PROMOTER VIA ChREBP AND SREBP-1

    PubMed Central

    Guha, Prajna; Aneja, Kawalpreet K.; Shilpi, Rasheda Y.; Haldar, Dipak

    2009-01-01

    We have recently identified two promoters, distal and proximal for rat mitochondrial glycerophosphate acyltransferase (mtGPAT). Here we are reporting further characterization of the promoters. Insulin and epidermal growth factor (EGF) stimulated while leptin and glucagon inhibited the luciferase activity of the distal promoter and the amounts of the distal transcript. Conversely, luciferase activity of the proximal promoter and proximal transcript remained unchanged due to these treatments. Only the distal promoter has binding sites for carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1 (SREBP-1). Electromobility shift assays and chromatin immunoprecipitation assays demonstrated that ChREBP and SREBP-1 bind to the mtGPAT distal promoter. Insulin and EGF increased while glucagon and leptin decreased the binding of SREBP-1 and ChREBP to the distal promoter. Thus, the distal promoter is the regulatory promoter while the proximal promoter acts constitutively for rat mtGPAT gene under the influence of hormones and growth factor. PMID:19682972

  14. Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis.

    PubMed

    Miyanaga, Akimasa; Iwasawa, Shohei; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2016-02-16

    Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein-protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK-ACP complexes. Because transient enzyme-ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK-ACP complexes, allowing the determination of the crystal structure of the VinK-VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK-VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT. PMID:26831085

  15. Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

    PubMed Central

    Miyanaga, Akimasa; Iwasawa, Shohei; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2016-01-01

    Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein–protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK–ACP complexes. Because transient enzyme–ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK–ACP complexes, allowing the determination of the crystal structure of the VinK–VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK–VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT. PMID:26831085

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

    PubMed

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

    2014-07-01

    Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production. PMID:24913048

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-10-01

    The lipase/acyltransferase from Candida parapsilosis (CpLIP2) immobilized on two synthetic resins (Accurel MP 1000 and Lewatit VP OC 1600) was used as catalyst for the production of biodiesel (fatty acid methyl esters, FAME) by transesterification of jatropha oil with methanol, in a lipid/aqueous system. The oil was dispersed in a buffer solution (pH 6.5) containing methanol in excess (2M in the biphasic system; molar ratio methanol/acyl chains 2:1). Transesterification was carried out at 30°C, under magnetic stirring, using 10% (w/w) of immobilized enzyme in relation to oil. The maximum FAME yields were attained after 8h reaction time: 80.5% and 93.8%, when CpLIP2 immobilized on Accurel MP 1000 or on Lewatit VP OC 1600 were used, respectively. CpLIP2 on both Accurel MP 1000 and Lewatit VP OC 1600 showed high operational stability along 5 consecutive 8h batches. PMID:27474957

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

    PubMed

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

    2015-03-01

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

  20. BAHD superfamily of acyl-CoA dependent acyltransferases in Populus and Arabidopsis: bioinformatics and gene expression

    SciTech Connect

    Yu, X.; Liu, C.

    2009-04-03

    Plant acyl-CoA dependent acyltransferases constitute a large specific protein superfamily, named BAHD. Using the conserved sequence motifs of BAHD members, we searched the genome sequences of Populus and Arabidopsis, and identified, respectively, 94- and 61-putative genes. Subsequently, we analyzed the phylogeny, gene structure, and chromosomal distribution of BAHD members of both species; then, we profiled expression patterns of BAHD genes by 'in silico' northern- and microarray-analyses based on public databases, and by RT-PCR. While our genomic- and bioinformatic- analyses provided full sets of BAHD superfamily genes, and cleaned up a few existing annotation errors, importantly it led to our recognizing several unique Arabidopsis BAHD genes that inversely overlapped with their neighboring genes on the genome, and disclosing a potential natural anti-sense regulation for gene expressions. Systemic gene-expression profiling of BAHD members revealed distinct tissue-specific/preferential expression patterns, indicating their diverse biological functions. Our study affords a strong knowledge base for understanding BAHD members evolutionary relationships and gene functions implicated in plant growth, development and metabolism.

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

    2016-01-01

    In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed “RU-SKI”) class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article “Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase” (Lanyon-Hogg et al., 2015) [1]. 1H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors. PMID:27077078

  2. Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

    PubMed Central

    Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

    2013-01-01

    Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl 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 seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

  3. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    PubMed

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  4. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    PubMed

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity.

  5. 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.

  6. Lecithin cholesterol acyltransferase deficiency protects from diet-induced insulin resistance and obesity--novel insights from mouse models.

    PubMed

    Ng, Dominic S

    2013-01-01

    Reduced plasma level of high-density lipoprotein cholesterol is an independent risk factor for atherosclerotic heart disease and is also a major diagnostic feature for the metabolic syndrome. Lecithin cholesterol acyltransferase (LCAT), an enzyme mediating the esterification of cholesterol in circulating lipoproteins, is one of the major modulators of high-density lipoprotein levels and composition. Loss-of-function mutations of LCAT invariably results in profound HDL deficiency and also modest hypertriglyceridemia (HTG). While intense effort has been devoted to investigate the role of LCAT in atherogenesis, which remains controversial, much less is known about whether LCAT also modulates glucose and energy homeostasis. In recent years, findings from studying the LCAT knockout mice began to suggest that LCAT deficiency, in spite of its unfavorable high triglyceride/low HDL lipid phenotypes, may confer protection from the development of insulin resistance and obesity. To date, alterations in specific metabolic pathways in liver, white adipose tissue, and skeletal muscle have been implicated. A better mechanistic understanding in the metabolic linkage between the primary biochemical action of LCAT and the downstream protective phenotypes will greatly facilitate the identification of potential novel pathways and targets in the treatment of obesity and diabetes. PMID:23374720

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

    PubMed

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

    2016-04-01

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

  8. Plasma lecithin:cholesterol acyltransferase and carotid intima-media thickness in European individuals at high cardiovascular risk

    PubMed Central

    Calabresi, Laura; Baldassarre, Damiano; Simonelli, Sara; Gomaraschi, Monica; Amato, Mauro; Castelnuovo, Samuela; Frigerio, Beatrice; Ravani, Alessio; Sansaro, Daniela; Kauhanen, Jussi; Rauramaa, Rainer; de Faire, Ulf; Hamsten, Anders; Smit, Andries J.; Mannarino, Elmo; Humphries, Steve E.; Giral, Philippe; Veglia, Fabrizio; Sirtori, Cesare R.; Franceschini, Guido; Tremoli, Elena

    2011-01-01

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. LCAT is a major factor in HDL remodeling and metabolism, and it has long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). The effect of LCAT on human atherogenesis is still controversial. In the present study, the plasma LCAT concentration was measured in all subjects (n = 540) not on drug treatment at the time of enrollment in the multicenter, longitudinal, observational IMPROVE study. Mean and maximum intima-media thickness (IMT) of the whole carotid tree was measured by B-mode ultrasonography in all subjects. In the entire cohort, LCAT quartiles were not associated with carotid mean and maximum IMT (P for trend 0.95 and 0.18, respectively), also after adjustment for age, gender, HDL-cholesterol (HDL-C), and triglycerides. No association between carotid IMT and LCAT quartiles was observed in men (P=0.30 and P=0.99 for mean and maximum IMT, respectively), whereas carotid IMT increased with LCAT quartiles in women (P for trend 0.14 and 0.019 for mean and maximum IMT, respectively). The present findings support the concept that LCAT is not required for an efficient reverse cholesterol transport and that a low plasma LCAT concentration and activity is not associated with increased atherosclerosis. PMID:21596929

  9. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

    SciTech Connect

    Kunst, L.; Somerville, C. ); Browse, J. )

    1989-07-01

    Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28{degree}C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.

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

    PubMed

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

    2016-02-01

    Statin drugs have proven a successful and relatively safe therapy for the treatment of atherosclerotic cardiovascular disease (CVD). However, even with the substantial low-density lipoprotein (LDL) cholesterol lowering achieved with statin treatment, CVD remains the top cause of death in developed countries. Selective inhibitors of the cholesterol esterifying enzyme sterol-O acyltransferase 2 (SOAT2) hold great promise as effective CVD therapeutics. In mouse models, previous work has demonstrated that either antisense oligonucleotide (ASO) or small molecule inhibitors of SOAT2 can effectively reduce CVD progression, and even promote regression of established CVD. Although it is well known that SOAT2-driven cholesterol esterification can alter both the packaging and retention of atherogenic apoB-containing lipoproteins, here we set out to determine whether SOAT2-driven cholesterol esterification can also impact basal and liver X receptor (LXR)-stimulated fecal neutral sterol loss. These studies demonstrate that SOAT2 is a negative regulator of LXR-stimulated fecal neutral sterol loss in mice. PMID:26729489

  11. Diacylglycerol and triacylglycerol as responses in a dual response surface-optimized process for diacylglycerol production by lipase-catalyzed esterification in a pilot packed-bed enzyme reactor.

    PubMed

    Lo, Seong-Koon; Cheong, Ling-Zhi; Arifin, Norlelawati; Tan, Chin-Ping; Long, Kamariah; Yusoff, Mohd Suria Affandi; Lai, Oi-Ming

    2007-07-11

    Diacylglycerol (DAG) and triacylglycerol (TAG) as responses on optimization of DAG production using a dual response approach of response surface methodology were investigated. This approach takes the molecular equilibrium of DAG into account and allows for the optimization of reaction conditions to achieve maximum DAG and minimum TAG yields. The esterification reaction was optimized with four factors using a central composite rotatable design. The following optimized conditions yielded 48 wt % DAG and 14 wt % TAG: reaction temperature of 66.29 degrees C, enzyme dosage of 4 wt %, fatty acid/glycerol molar ratio of 2.14, and reaction time of 4.14 h. Similar results were achieved when the process was scaled up to a 10 kg production in a pilot packed-bed enzyme reactor. Lipozyme RM IM did not show any significant activity losses or changes in fatty acid selectivity on DAG synthesis during the 10 pilot productions. However, lipozyme RM IM displayed higher selectivity toward the production of oleic acid-enriched DAG. The purity of DAG oil after purification was 92 wt %.

  12. Comparison of alkylacylglycerol vs. diacylglycerol as activators of mitogen-activated protein kinase and cytosolic phospholipase A2 in human neutrophil priming.

    PubMed

    Nixon, A B; Seeds, M C; Bass, D A; Smitherman, P K; O'Flaherty, J T; Daniel, L W; Wykle, R L

    1997-08-16

    In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.

  13. Glycidol exposure evaluation of humans who have ingested diacylglycerol oil containing glycidol fatty acid esters using hemoglobin adducts.

    PubMed

    Honda, Hiroshi; Fujii, Kenkichi; Yamaguchi, Tohru; Ikeda, Naohiro; Nishiyama, Naohiro; Kasamatsu, Toshio

    2012-11-01

    Glycidol fatty acid esters (GEs) have been found as impurities in refined edible oils including diacylglycerol (DAG) oil, and concerns of possible exposure to glycidol (G), a known animal carcinogen, during digestion have been raised. We previously measured N-(2,3-dihydroxy-propyl)valine (diHOPrVal), a G hemoglobin adduct, for DAG oil exposed and non-exposed groups and showed there was no significant difference between them. In the present study, we conducted an additional analysis to verify the outcome of the previous report. The first experiment was designed as a matched case-control study to adjust variables with an increased sample size. The average levels of diHOPrVal were 6.9 pmol/g-globin (95%CI: 4.9-9.0) for 14 DAG oil exposed subjects and 7.3 pmol/g-globin (95%CI: 6.1-8.5) for 42 non-exposed volunteers, and no significant difference in levels was found between the two groups. In a second experiment, we compared the adduct levels of 12 DAG oil exposed subjects before and after discontinuing use of DAG oil, and found there was no significant change in diHOPrVal levels (from 7.1±1.1 to 7.5±1.4 pmol/g-globin). These results suggest that there was no increased exposure to G for humans who ingested DAG oil daily, although the evaluated population was limited.

  14. The CDP-Ethanolamine Pathway Regulates Skeletal Muscle Diacylglycerol Content and Mitochondrial Biogenesis without Altering Insulin Sensitivity.

    PubMed

    Selathurai, Ahrathy; Kowalski, Greg M; Burch, Micah L; Sepulveda, Patricio; Risis, Steve; Lee-Young, Robert S; Lamon, Severine; Meikle, Peter J; Genders, Amanda J; McGee, Sean L; Watt, Matthew J; Russell, Aaron P; Frank, Matthew; Jackowski, Suzanne; Febbraio, Mark A; Bruce, Clinton R

    2015-05-01

    Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP:phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis. PMID:25955207

  15. Self-Assembly and Lipid Interactions of Diacylglycerol Lactone Derivatives Studied at the Air/Water Interface

    PubMed Central

    Philosof-Mazor, Liron; Volinsky, Roman; Comin, Maria J.; Lewin, Nancy E.; Kedei, Noemi; Blumberg, Peter M.; Marquez, Victor E.; Jelinek, Raz

    2009-01-01

    Synthetic diacylglycerol lactones (DAG-lactones) have been shown to be effective modulators of critical cellular signaling pathways. The biological activity of these amphiphilic molecules depends in part upon their lipid interactions within the cellular plasma membrane. This study explores the thermodynamic and structural features of DAG-lactone derivatives and their lipid interactions at the air/water interface. Surface-pressure/area isotherms and Brewster angle microscopy revealed the significance of specific side-groups attached to the terminus of a very rigid 4-(2-phenylethynyl) benzoyl chain of the DAG-lactones, which affected both the self-assembly of the molecules and their interactions with phospholipids. The experimental data highlight the formation of different phases within mixed DAG-lactone/phospholipid monolayers and underscore the relationship between the two components in binary mixtures of different mole ratios. Importantly, the results suggest that DAG-lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases that form, for example, by cholesterol. Moreover, the size and charge of the phospholipid headgroups do not seem to affect DAG-lactone interactions with lipids. PMID:18788772

  16. Host Lipid and Temperature as Important Screening Variables for Crystallizing Integral Membrane Proteins in Lipidic Mesophases. Trials with Diacylglycerol Kinase

    PubMed Central

    Li, Dianfan; Shah, Syed T. A.; Caffrey, Martin

    2013-01-01

    A systematic study of the crystallization of an α-helical, integral membrane enzyme, diacylglycerol kinase, DgkA, using the lipidic cubic mesophase or in meso method is described. These trials have resulted in the production of blocky, rhombohedron-shaped crystals of diffraction quality currently in use for structure determination. Dramatic improvements in crystal quality were obtained when the identity of the lipid used to form the mesophase bilayer into which the protein was reconstituted as a prelude to crystallogenesis was varied. These monoacylglycerol lipids incorporated fatty acyl chains ranging from 14 to 18 carbon atoms long with cis olefinic bonds located toward the middle of the chain. Best crystals were obtained with a lipid that had an acyl chain 15 carbon atoms long with the double bond between carbons 7 and 8. It is speculated that the effectiveness of this lipid derives from hydrophobic mismatch between the target integral membrane protein and the bilayer of the host mesophase. Low temperature (4 °C) worked in concert with the short chain lipid to provide high quality crystals. Recommended screening strategies for crystallizing membrane proteins that include host lipid type and low temperature are made on the basis of this and related in meso crystallization trials. PMID:23956688

  17. Physicochemical properties of peanut oil-based diacylglycerol and their derived oil-in-water emulsions stabilized by sodium caseinate.

    PubMed

    Long, Zhao; Zhao, Mouming; Liu, Ning; Liu, Daolin; Sun-Waterhouse, Dongxiao; Zhao, Qiangzhong

    2015-10-01

    High purity peanut oil-based diacylglycerol (PO-DAG) (94.95 wt%) was prepared via enzymatic glycerolysis from peanut oil (PO). The resulting dominance of DAGs was proven to greatly influence the properties of corresponding fresh or frozen-thawed emulsions. Stable fresh oil-in-water emulsions were produced using either PO-DAG or PO, with stability enhanced by increased concentrations of Na-CN. The lower equilibrium interfacial tension along with greater negative ζ-potential of PO revealed that Na-CN was preferentially adsorbed to the PO interface. Adding 0.05 mol/L NaCl to the PO emulsions minimized depletion flocculation caused by the unadsorbed Na-CN, but further NaCl addition increased oil droplet size and concomitant coalescence. For the PO-DAG emulsions, adding 0.2 mol/L NaCl did not significantly (p>0.05) affect their ζ-potential but adding 0.05 or 0.1 mol/L NaCl lowered ζ-potential, although NaCl at these concentrations increased oil droplet size and coalescence. Freezing-thawing process considerably weakened the stability of PO-DAG emulsions.

  18. Energy value and digestibility of dietary oil containing mainly 1,3-diacylglycerol are similar to those of triacylglycerol.

    PubMed

    Taguchi, H; Nagao, T; Watanabe, H; Onizawa, K; Matsuo, N; Tokimitsu, I; Itakura, H

    2001-04-01

    Diacylglycerol (DAG) is a component of various vegetable oils. Approximately 70% of the DAG in edible oils are in the configuration of 1,3-DAG. We recently showed that long-term ingestion of dietary oil containing mainly 1,3-DAG reduces body fat accumulation in humans as compared to triacylglycerol (TAG) oil with a similar fatty acid composition. As the first step to elucidate the mechanism for this result, we examined the difference in the bioavailabilities of both oils by measuring food energy values and digestibilities in rats. Energy values of the DAG oil and the TAG oil, measured by bomb calorimeter, were 38.9 and 39.6 kJ/g, respectively. Apparent digestibility expressed according to the formula: (absorbed) x (ingested)(-1) x 100 = (ingested - excreted in feces) x (ingested)(-1) x 100 for the DAG oil and the TAG oil were 96.3+/-0.4 and 96.3+/-0.3% (mean +/- SEM), respectively. The similarity in the bioavailabilities of both oils supports the hypothesis that the reduced fat accumulation by dietary DAG is caused by the different metabolic fates after the absorption into the gastrointestinal epithelial cells. PMID:11383689

  19. Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

    PubMed Central

    van Blitterswijk, W J; Hilkmann, H

    1993-01-01

    Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation. Images PMID:8392931

  20. Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase immobilised on nano-sized magnetite particles.

    PubMed

    Meng, Xiao; Xu, Gang; Zhou, Qin-Li; Wu, Jian-Ping; Yang, Li-Rong

    2014-01-15

    Recently, 1,3-DAGs (1,3-diacylglycerols) have attracted considerable attention as healthy components of food, oil and pharmaceutical intermediates. Generally, 1,3-DAG is prepared by lipase-mediated catalysis in a solvent free system. However, the system's high reaction temperature (required to reach the reactants' melting point), high substrate concentration and high viscosity severely reduce the lipase's activity, selectivity and recycling efficiency. In this report, MjL (Mucor javanicus lipase) was found to have the best performance in the solvent-free synthesis of 1,3-DAGs of several common commercial lipases. By covalent binding to amino-group-activated NSM (nano-sized magnetite) particles and cross-linking to form an enzyme aggregate coat, MjL's specific activity increased 10-fold, and was able to be reused for 10 cycles with 90% residual activity at 55°C. 1,3-DAGs of lauric, myristic, palmitic, stearic, oleic and linoleic acid were prepared using the resulting immobilised enzyme, all with yields greater than 90%, and the reaction time was also greatly reduced. PMID:24054246

  1. Suppressed histamine release from rat peritoneal mast cells by ultraviolet B irradiation: decreased diacylglycerol formation as a possible mechanism

    SciTech Connect

    Danno, K.; Fujii, K.; Tachibana, T.; Toda, K.; Horio, T.

    1988-06-01

    This study was designed to investigate the effect of ultraviolet B (UVB) irradiation on mast cell functions. Purified mast cells obtained from rat peritoneal cavity were irradiated with UVB and subsequently exposed to a degranulator, compound 48/80, or the calcium ionophore A-23187. The amount of histamine released from mast cells measured by the enzyme isotopic assay was significantly decreased by UVB irradiation (100-400 mJ/cm2). Within this dose range, UVB alone was not cytotoxic to the cells because it did not induce histamine release. The suppression was observed when mast cells were subjected to degranulation without intervals after UVB irradiation, and even after 5 h postirradiation. The wavelength of 300 nm from a monochromatic light source showed the maximum effect. When mast cells prelabeled with (/sup 3/H)arachidonate were irradiated and challenged by compound 48/80, label accumulation in diacylglycerol produced by the phosphatidylinositol cycle was considerably decreased by UVB irradiation. From these results, we hypothesize that, within an adequate irradiation dose, UVB irradiation suppresses histamine release from mast cells, probably by causing noncytotoxic damage to the membrane phospholipid metabolism, which is tied to the degranulation mechanisms.

  2. Downregulation of diacylglycerol kinase ζ enhances activation of cytokine-induced NF-κB signaling pathway.

    PubMed

    Tsuchiya, Rieko; Tanaka, Toshiaki; Hozumi, Yasukazu; Nakano, Tomoyuki; Okada, Masashi; Topham, Matthew K; Iino, Mitsuyoshi; Goto, Kaoru

    2015-02-01

    The transcription factor NF-κB family serves as a key component of many pathophysiological events such as innate and adaptive immune response, inflammation, apoptosis, and oncogenesis. Various cell signals trigger activation of the regulatory mechanisms of NF-κB, resulting in its nuclear translocation and transcriptional initiation. The diacylglycerol kinase (DGK) family, a lipid second messenger-metabolizing enzyme in phosphoinositide signaling, is shown to regulate widely various cellular processes. Results of recent studies suggest that one family member, DGKζ, is closely involved in immune and inflammatory responses. Nevertheless, little is known about the regulatory mechanism of DGKζ on NF-κB pathway in cytokine-induced inflammatory signaling. This study shows that siRNA-mediated DGKζ knockdown in HeLa cells facilitates degradation of IκB, followed by nuclear translocation of NF-κB p65 subunit. In addition, DGKζ-deficient MEFs show upregulation of p65 subunit phosphorylation at Serine 468 and 536 and its interaction with CBP transcriptional coactivator upon TNF-α stimulation. These modifications of p65 subunit might engender enhanced NF-κB transcriptional reporter assay of DGKζ knockdown cells. These findings provide further insight into the regulatory mechanisms of cytokine-induced NF-κB activation.

  3. CDP-diacylglycerol synthetase coordinates cell growth and fat storage through phosphatidylinositol metabolism and the insulin pathway.

    PubMed

    Liu, Yuan; Wang, Wei; Shui, Guanghou; Huang, Xun

    2014-03-01

    During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.

  4. Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

    PubMed

    Chen, Chun-Yen; Kao, Ai-Ling; Tsai, Zheng-Chia; Chow, Te-Jin; Chang, Hsin-Yueh; Zhao, Xin-Qing; Chen, Po-Ting; Su, Hsiang-Yen; Chang, Jo-Shu

    2016-03-01

    Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production.

  5. Expression and localization of type II diacylglycerol kinase isozymes δ and η in the developing mouse brain.

    PubMed

    Usuki, Takako; Sakai, Hiromichi; Shionoya, Takao; Sato, Naruki; Sakane, Fumio

    2015-01-01

    The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II-VI of the cerebral cortex; CA-CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II-VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.

  6. Highly efficient solvent-free synthesis of 1,3-diacylglycerols by lipase immobilised on nano-sized magnetite particles.

    PubMed

    Meng, Xiao; Xu, Gang; Zhou, Qin-Li; Wu, Jian-Ping; Yang, Li-Rong

    2014-01-15

    Recently, 1,3-DAGs (1,3-diacylglycerols) have attracted considerable attention as healthy components of food, oil and pharmaceutical intermediates. Generally, 1,3-DAG is prepared by lipase-mediated catalysis in a solvent free system. However, the system's high reaction temperature (required to reach the reactants' melting point), high substrate concentration and high viscosity severely reduce the lipase's activity, selectivity and recycling efficiency. In this report, MjL (Mucor javanicus lipase) was found to have the best performance in the solvent-free synthesis of 1,3-DAGs of several common commercial lipases. By covalent binding to amino-group-activated NSM (nano-sized magnetite) particles and cross-linking to form an enzyme aggregate coat, MjL's specific activity increased 10-fold, and was able to be reused for 10 cycles with 90% residual activity at 55°C. 1,3-DAGs of lauric, myristic, palmitic, stearic, oleic and linoleic acid were prepared using the resulting immobilised enzyme, all with yields greater than 90%, and the reaction time was also greatly reduced.

  7. Digestion of the 1-O-alkyl diacylglycerol ethers of Atlantic dogfish liver oils by Atlantic salmon Salmo salar.

    PubMed

    Kang, S J; Lall, S P; Ackman, R G

    1997-01-01

    Dogfish (Squalus acanthias) liver poses a waste disposal problem in Canada because it is not utilized for any commercial purpose. The liver of Atlantic dogfish, which is often up to 20% of the weight of the fish, contains 40-70% oil. The oil contains about 30-40% 1-O-alkyl diacylglycerol ethers (DAGE) which render it unacceptable for human use, and it has also not been considered satisfactory for animal feed use. Polyunsaturated fatty acids (20:5n-3 and 22:6n-3) are present in dogfish liver oils at levels comparable to those in herring oil. Dogfish liver oil could be a source of essential fatty acids for Atlantic salmon (Salmo salar), but their ability to hydrolyze DAGE from dogfish oil has not been examined. Experiments were designed to measure the digestibility of fatty acids of DAGE in salmon. The fatty acid moieties were liberated by the digestive enzymes of the fish and made readily available as a source of energy. The 1-O-alkylglycerol ether moiety was absorbed to a small extent but should not constitute a health problem in either the fish or the human fish consumer. The long-chain polyunsaturated fatty acids were particularly well absorbed, with an apparent digestibility in salmon of 87-95% when feeding on dogfish liver oil. The total fatty acids and other lipids were in fact both absorbed to the extent of approximately 85%.

  8. Anti-Inflammatory Potential of Monogalactosyl Diacylglycerols and a Monoacylglycerol from the Edible Brown Seaweed Fucus spiralis Linnaeus

    PubMed Central

    Lopes, Graciliana; Daletos, Georgios; Proksch, Peter; Andrade, Paula B.; Valentão, Patrícia

    2014-01-01

    A monoacylglycerol (1) and a 1:1 mixture of two monogalactosyl diacylglycerols (MGDGs) (2 and 3) were isolated from the brown seaweed Fucus spiralis Linnaeus. The structures were elucidated by spectroscopic means (NMR and MS) and by comparison with the literature. Compound 1 was composed of a glycerol moiety linked to oleic acid (C18:1 Ω9). Compounds 2 and 3 contained a glycerol moiety linked to a galactose unit and eicosapentaenoic acid (C20:5 Ω3) combined with octadecatetraenoic acid (C18:4 Ω3) or linolenic acid (C18:3 Ω3), respectively. The isolated compounds were tested for their cytotoxic and anti-inflammatory activity in RAW 264.7 macrophage cells. All of them inhibited NO production at non-cytotoxic concentrations. The fraction consisting of compounds 2 and 3, in a ratio of 1:1, was slightly more effective than compound 1 (IC50 of 60.06 and 65.70 µg/mL, respectively). To our knowledge, this is the first report of these compounds from F. spiralis and on their anti-inflammatory capacity. PMID:24619274

  9. Immobilization of Candida antarctica lipase B onto SBA-15 and their application in glycerolysis for diacylglycerols synthesis.

    PubMed

    Cai, Chunsheng; Gao, Yongqing; Liu, Yan; Zhong, Nanjing; Liu, Ning

    2016-12-01

    In this study, Candida antarctica lipase B (CALB) was immobilized on SBA-15 with three pore diameters. CALB loading was found increased with CALB concentration increasing from 20.3 to 80.12μg/ml. Higher CALB loading was observed from SBA-15 with pore diameters at 8.1nm (SBA-15(8.1)), yet highest hydrolytic activity was found at SBA-15(12.5). Thermal stability of the immobilized CALB (SBA-15-CALB) samples was greatly influenced by their water content, after 4h storage at 70°C, 8.93 and 67.4% of the initial activity was observed from SBA-15-CALB samples with water content at 9.23 and 3.22% respectively. The SBA-15-CALB samples were then used in glycerolysis of corn oil, and 53.6wt% of diacylglycerols was obtained after optimization. The operational stability was tested, and after 5 consecutive applications, 92.5 and 80.3% of the initial glycerolysis activity was remained respectively from SBA-15(6.6)-CALB and SBA-15(12.5)-CALB. PMID:27374525

  10. 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

  11. 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

  12. Genetic Evidence for the Reduction of Brassinosteroid Levels by a BAHD Acyltransferase-Like Protein in Arabidopsis1[W][OA

    PubMed Central

    Roh, Hyungmin; Jeong, Cheol Woong; Fujioka, Shozo; Kim, Youn Kyung; Lee, Sookjin; Ahn, Ji Hoon; Do Choi, Yang; Lee, Jong Seob

    2012-01-01

    Brassinosteroids (BRs) are a group of steroidal hormones involved in plant development. Although the BR biosynthesis pathways are well characterized, the BR inactivation process, which contributes to