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Sample records for acyltransferase lcat activity

  1. A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity.

    PubMed Central

    Funke, H; von Eckardstein, A; Pritchard, P H; Albers, J J; Kastelein, J J; Droste, C; Assmann, G

    1991-01-01

    Epidemiological as well as biochemical evidence of recent years has established that a low plasma level of high density lipoprotein-cholesterol is a predictor for the risk of coronary artery disease. However, there is a heterogeneous group of rare familial disorders, characterized by severe high density lipoprotein deficiency, in which the predicted increased risk is not clearly apparent. One such disorder has been called fish eye disease to reflect the massive corneal opacification seen in these patients. In this report, we describe the biochemical and genetic presentation of two German fish eye disease homozygotes and their family members. Vertical transmission of a decrease in the specific activity of lecithin-cholesterol acyltransferase (EC 2.3.1.43) indicated that this enzyme was a candidate gene for harboring the defect responsible for this disorder. Direct sequencing of DNA segments amplified by the polymerase chain reaction (PCR) that encode the exons of the lecithin-cholesterol acyltransferase gene led to the identification of a homozygous mutation resulting in the substitution of threonine at codon 123 for an isoleucine residue in both individuals. Family analysis in an extended pedigree was used to establish a causal relationship between this mutation and the biochemical phenotype for fish eye disease. The homozygous presence of this mutation in two phenotypically homozygous members of an unrelated Dutch family with fish eye disease further supports this finding. Images PMID:2052566

  2. Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner.

    PubMed

    Nesan, Dinushan; Tavallaee, Ghazaleh; Koh, Deborah; Bashiri, Amir; Abdin, Rawand; Ng, Dominic S

    2015-12-18

    Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr(-/-)xLcat(-/-) or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr(-/-)xLcat(+/+) single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr(+/+)xLcat(-/-) (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.

  3. Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

    PubMed

    Vickaryous, Nicola K; Teh, Evelyn M; Stewart, Bruce; Dolphin, Peter J; Too, Catherine K L; McLeod, Roger S

    2003-03-21

    Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the

  4. 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.

  5. Serum lipoprotein composition, lecithin cholesterol acyltransferase and tissue lipase activities in pregnant diabetic rats and their offspring receiving enriched n-3 PUFA diet.

    PubMed

    Soulimane-Mokhtari, N A; Guermouche, B; Saker, M; Merzouk, S; Merzouk, H; Hichami, A; Madani, S; Khan, N A; Prost, J

    2008-03-01

    The effects of dietary n-3 polyunsaturated fatty acids on lipoprotein concentrations and on lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL) and lecithin cholesterol acyltransferase (LCAT) activities were studied in streptozotocin-induced diabetic rats during pregnancy and in their macrosomic offspring from birth to adulthood. Pregnant diabetic and control rats were fed Isio-4 diet (vegetable oil) or EPAX diet (concentrated marine omega-3 EPA/DHA oil), the same diets were consumed by pups at weaning. Compared with control rats, diabetic rats showed, during pregnancy, a significant elevation in very low density lipoprotein (VLDL) and low and high density lipoprotein (LDL-HDL(1))-triglyceride, cholesterol and apoprotein B100 concentrations and a reduction in apoprotein A-I levels. HTGL activity was high while LPL and LCAT activities were low in these rats. The macrosomic pups of Isio-4-fed diabetic rats showed a significant enhancement in triglyceride and cholesterol levels at birth and during adulthood with a concomitant increase in lipase and LCAT activities. EPAX diet induces a significant diminution of VLDL and LDL-HDL(1) in mothers and in their macrosomic pups, accompanied by an increase in cholesterol and apoprotein A-I levels in HDL(2-3) fraction. It also restores LPL, HTGL and LCAT activities to normal range. EPAX diet ameliorates considerably lipoprotein disorders in diabetic mothers and in their macrosomic offspring.

  6. Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation.

    PubMed

    Hine, David; Mackness, Bharti; Mackness, Mike

    2012-02-01

    The inhibition of low-density lipoprotein (LDL) oxidation by high-density lipoprotein (HDL) is a major antiatherogenic property of this lipoprotein. This activity is due, in part, to HDL associated proteins. However, whether these proteins interact in the antioxidant activity of HDL is unknown. LDL was incubated with apolipoprotein A1 (apo A1), lecithin:cholesterol acyltransferase (LCAT), and paraoxonase-1 (PON1) alone or in combination, in the presence or absence of HDL under oxidizing conditions. LDL lipid peroxide concentrations were determined. Apo A1, LCAT, and PON1 all inhibit LDL oxidation in the absence of HDL and enhance the ability of HDL to inhibit LDL oxidation. Their effect was additive rather than synergistic; the combination of these proteins significantly enhanced the length of time LDL was protected from oxidation. This seemed to be due to the ability of PON1 to prevent the oxidative inactivation of LCAT. Apo A1, LCAT, and PON1 can all contribute to the antioxidant activity of HDL in vitro. The combination of apo A1, LCAT, and PON1 prolongs the time that HDL can prevent LDL oxidation, due, at least in part, to the prevention LCAT inactivation.

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

    PubMed Central

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

    2016-01-01

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

  8. 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.

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

    PubMed

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

    1978-01-01

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

  10. Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

    PubMed

    Bielicki, J K; Knoff, L J; Tribble, D L; Forte, T M

    2001-03-01

    In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion

  11. Increased activity of lecithin:cholesterol acyltransferase during short-term oral estrogen progestin replacement therapy in a group of postmenopausal women.

    PubMed

    Ulloa, N; Verdugo, C; Rios, M; Sepúlveda, J; Sepúlveda, S; Naveas, R; Calvo, C

    1998-03-01

    The aim of the study was to assess the short-term effect of estrogen-progestin therapy on the plasma level of lecithin: cholesterol acyltransferase ([LCAT] EC 2.3.1.43), a key enzyme in the cholesterol reverse-transport process. The trial included 21 women with at least 6 months of menopause, which was confirmed by anamnesis, physical evaluation, and follicle-stimulating hormone (FSH) determination. Women receiving pharmacological treatment or who had any kind of endocrine disorder were excluded. In addition, we evaluated and confirmed normal Papanicolaou and mammography tests in all 21 women included in the trial. They received conjugated equine estrogen 0.625 mg daily, plus cyclic medroxyprogesterone acetate (5 mg daily) for 12 days each month. Plasma levels of LCAT, cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, apoB, and apoAI were evaluated before and after 1 and 3 months of therapy. Pretherapy and posttherapy results were analyzed statistically by Wilcoxon's rank-sum test for paired samples. No significant changes were observed either for body mass index or for blood pressure. A significant increase in plasma LCAT activity was found at the first and third month posttherapy (P < .005). In addition, after 3 months of therapy, HDL-C significantly increased (P < .005), in contrast to the significant decrease detected in total cholesterol (P < .025), LDL-C (P < .005), cholesterol to HDL-C and LDL-C/HDL-C ratios (P < .005). Triglyceride levels did not show significant modification. In conclusion, our results indicate that short-term estrogen-progestin therapy produces a significant increase in plasma LCAT activity, as well as beneficial changes in the lipid profile, in postmenopausal women.

  12. 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.

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

    PubMed

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

    2016-01-14

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

  14. ACAT inhibition reverses LCAT deficiency and improves plasma HDL in chronic renal failure.

    PubMed

    Vaziri, N D; Liang, K

    2004-11-01

    Chronic renal failure (CRF) is associated with increased risk of arteriosclerotic cardiovascular disease and profound alteration of plasma lipid profile. Uremic dyslipidemia is marked by increased plasma concentration of ApoB-containing lipoproteins and impaired high-density lipoprotein (HDL)-mediated reverse cholesterol transport. These abnormalities are, in part, due to acquired LCAT deficiency and upregulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT). ACAT catalyzes intracellular esterification of cholesterol, thereby promoting hepatic production of ApoB-containing lipoproteins and constraining HDL-mediated cholesterol uptake in the peripheral tissues. In view of the above considerations, we tested the hypothesis that pharmacological inhibition of ACAT may ameliorate CRF-induced dyslipidemia. 5/6 Nephrectomized rats were treated with either ACAT inhibitor IC-976 (30 mg.kg(-1).day(-1)) or placebo for 6 wk. Sham-operated rats served as controls. Key cholesterol-regulating enzymes, plasma lipids, and creatinine clearance were measured. The untreated CRF rats exhibited increased plasma low-density lipoprotein (LDL) and very LDL (VLDL) cholesterol, unchanged plasma HDL cholesterol, elevated total cholesterol-to-HDL cholesterol ratio, reduced liver microsomal free cholesterol, and diminished creatinine clearance. This was accompanied by reduced plasma LCAT, increased hepatic ACAT-2 mRNA, ACAT-2 protein and ACAT activity, and unchanged hepatic HMG-CoA reductase and cholesterol 7alpha-hydroxylase. ACAT inhibitor raised plasma HDL cholesterol, lowered LDL and VLDL cholesterol, and normalized total cholesterol-to-HDL cholesterol ratio without changing total cholesterol concentration (hence, a shift from ApoB-containing lipoproteins to HDL). This was accompanied by normalizations of hepatic ACAT activity and plasma LCAT. In conclusion, inhibition of ACAT reversed LCAT deficiency and improved plasma HDL level in CRF rats. Future studies are needed to explore

  15. 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

  16. 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.

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

    PubMed Central

    Schlossman, D M; Bell, R M

    1978-01-01

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

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

    USGS Publications Warehouse

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

    2008-01-01

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

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

    PubMed

    Pavlovic, Nikola M; Orem, William H; Tatu, Calin A; Lerch, Harry E; Bunnell, Joseph E; Feder, Gerald L; Kostic, Emina N; Ordodi, Valentin L

    2008-03-01

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

  20. Lipoprotein X Causes Renal Disease in LCAT Deficiency.

    PubMed

    Ossoli, Alice; Neufeld, Edward B; Thacker, Seth G; Vaisman, Boris; Pryor, Milton; Freeman, Lita A; Brantner, Christine A; Baranova, Irina; Francone, Nicolás O; Demosky, Stephen J; Vitali, Cecilia; Locatelli, Monica; Abbate, Mauro; Zoja, Carlamaria; Franceschini, Guido; Calabresi, Laura; Remaley, Alan T

    2016-01-01

    Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

  1. Lipoprotein X Causes Renal Disease in LCAT Deficiency

    PubMed Central

    Thacker, Seth G.; Vaisman, Boris; Pryor, Milton; Freeman, Lita A.; Brantner, Christine A.; Baranova, Irina; Francone, Nicolás O.; Demosky, Stephen J.; Vitali, Cecilia; Locatelli, Monica; Abbate, Mauro; Zoja, Carlamaria; Franceschini, Guido; Calabresi, Laura; Remaley, Alan T.

    2016-01-01

    Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis. PMID:26919698

  2. Binding and functional effects of transcription factors Sp1 and Sp3 on the proximal human lecithin:cholesterol acyltransferase promoter.

    PubMed

    Hoppe, K L; Francone, O L

    1998-05-01

    Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5' variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA-protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of transactivating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.

  3. 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.

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

    PubMed Central

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

    2015-01-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high resolution crystal structures of human LPLA2 and a low resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome. PMID:25727495

  5. Lecithin:Cholesterol Acyltransferase: From Biochemistry to Role in Cardiovascular Disease

    PubMed Central

    Rousset, Xavier; Vaisman, Boris; Amar, Marcelo; Sethi, Amar A.; Remaley, Alan T.

    2010-01-01

    Purpose of review We discuss the latest findings on the biochemistry of lecithin:cholesterol acyltransferase (LCAT), the effect of LCAT on atherosclerosis, clinical features of LCAT deficiency, and the impact of LCAT on cardiovascular disease from human studies. Recent findings Although there has been much recent progress in the biochemistry of LCAT and its effect on HDL metabolism, its role in the pathogenesis of atherosclerosis is still not fully understood. Studies from various animal models have revealed a complex interaction between LCAT and atherosclerosis that may be modified by diet and by other proteins that modify lipoproteins. Furthermore, the ability of LCAT to lower apoB appears to be the best way to predict its effect on atherosclerosis in animal models. Recent studies on patients with LCAT deficiency have shown a modest but significant increase incidence of cardiovascular disease consistent with a beneficial effect of LCAT on atherosclerosis. The role of LCAT in the general population, however, have not revealed a consistent association with cardiovascular disease. Summary Recent research findings from animal and humans studies have revealed a potential beneficial role of LCAT in reducing atherosclerosis but additional studies are necessary to better establish the linkage between LCAT and cardiovascular disease. PMID:19306528

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

    PubMed

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

    2003-11-28

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

  7. A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein.

    PubMed

    Gu, Xiaodong; Wu, Zhiping; Huang, Ying; Wagner, Matthew A; Baleanu-Gogonea, Camelia; Mehl, Ryan A; Buffa, Jennifer A; DiDonato, Anthony J; Hazen, Leah B; Fox, Paul L; Gogonea, Valentin; Parks, John S; DiDonato, Joseph A; Hazen, Stanley L

    2016-03-18

    The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu(159)-Leu(170)) in nascent HDL, the so-called "solar flare" (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861-868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro(165), Tyr(166), Ser(167), and Asp(168)) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr(166) was further supported using reconstituted HDL generated from apoA-I mutants (Tyr(166) → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr(166)-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr(166)-apoA-I, after subcutaneous injection into hLCAT(Tg/Tg), apoA-I(-/-) mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency.

  8. A Systematic Investigation of Structure/Function Requirements for the Apolipoprotein A-I/Lecithin Cholesterol Acyltransferase Interaction Loop of High-density Lipoprotein*

    PubMed Central

    Gu, Xiaodong; Wu, Zhiping; Huang, Ying; Wagner, Matthew A.; Baleanu-Gogonea, Camelia; Mehl, Ryan A.; Buffa, Jennifer A.; DiDonato, Anthony J.; Hazen, Leah B.; Fox, Paul L.; Gogonea, Valentin; Parks, John S.; DiDonato, Joseph A.; Hazen, Stanley L.

    2016-01-01

    The interaction of lecithin-cholesterol acyltransferase (LCAT) with apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) maturation. We previously identified a highly solvent-exposed apoA-I loop domain (Leu159–Leu170) in nascent HDL, the so-called “solar flare” (SF) region, and proposed that it serves as an LCAT docking site (Wu, Z., Wagner, M. A., Zheng, L., Parks, J. S., Shy, J. M., 3rd, Smith, J. D., Gogonea, V., and Hazen, S. L. (2007) Nat. Struct. Mol. Biol. 14, 861–868). The stability and role of the SF domain of apoA-I in supporting HDL binding and activation of LCAT are debated. Here we show by site-directed mutagenesis that multiple residues within the SF region (Pro165, Tyr166, Ser167, and Asp168) of apoA-I are critical for both LCAT binding to HDL and LCAT catalytic efficiency. The critical role for possible hydrogen bond interaction at apoA-I Tyr166 was further supported using reconstituted HDL generated from apoA-I mutants (Tyr166 → Glu or Asn), which showed preservation in both LCAT binding affinity and catalytic efficiency. Moreover, the in vivo functional significance of NO2-Tyr166-apoA-I, a specific post-translational modification on apoA-I that is abundant within human atherosclerotic plaque, was further investigated by using the recombinant protein generated from E. coli containing a mutated orthogonal tRNA synthetase/tRNACUA pair enabling site-specific insertion of the unnatural amino acid into apoA-I. NO2-Tyr166-apoA-I, after subcutaneous injection into hLCATTg/Tg, apoA-I−/− mice, showed impaired LCAT activation in vivo, with significant reduction in HDL cholesteryl ester formation. The present results thus identify multiple structural features within the solvent-exposed SF region of apoA-I of nascent HDL essential for optimal LCAT binding and catalytic efficiency. PMID:26797122

  9. A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

    Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.

  10. Activation of lecithin cholesterol acyltransferase by human apolipoprotein E in discoidal complexes with lipids.

    PubMed

    Zorich, N; Jonas, A; Pownall, H J

    1985-07-25

    In a continued investigation of lecithin cholesterol acyltransferase reaction with micellar discoidal complexes of phosphatidylcholine, cholesterol, and various water soluble apolipoproteins, we prepared complexes containing human apo-E by the cholate dialysis method. These complexes were systematically compared to apo-A-I complexes synthesized under the same reaction conditions. Apo-E complexes (134 A in diameter) were slightly larger than apo-A-I complexes (110 A) but were very similar in terms of their protein and lipid content (2.4:0.10:1.0, egg phosphatidylcholine/cholesterol/apolipoprotein, w/w) and in the percentage of apolipoprotein in alpha-helical structure (72-74%). Concentration and temperature-dependence experiments on the velocity of the lecithin cholesterol acyltransferase reaction revealed differences in apparent Km values and small differences in apparent Vmax but very similar activation energies (18-20 kcal/mol). These observations suggest that differences in lecithin cholesterol acyltransferase activation by apo-A-I and apo-E are primarily a result of different affinities of the enzyme for the particles but that the rate-limiting step of the reaction is comparable for both complexes. Apo-E was found to be 18% as effective as apo-A-I in activating purified human lecithin cholesterol acyltransferase. Addition of free apo-A-I to apo-E complexes resulted in the exchange of bound for free apolipoprotein causing a slight increase in the reactivity with the enzyme when the incubation mixture was assayed. When the unbound apolipoproteins were removed by ultracentrifugation reisolated complexes containing both apo-E and apo-A-I demonstrated an even greater increase in reactivity with the enzyme.

  11. Genetics Home Reference: complete LCAT deficiency

    MedlinePlus

    ... levels of HDL cholesterol and atherosclerosis, a variable relationship--a review of LCAT deficiency. Vasc Health Risk ... healthcare professional . About Genetics Home Reference Site Map Customer Support Selection Criteria for Links USA.gov Copyright ...

  12. Activities of acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) in microsomal preparations of developing sunflower and safflower seeds.

    PubMed

    Banaś, Walentyna; Sanchez Garcia, Alicia; Banaś, Antoni; Stymne, Sten

    2013-06-01

    The last step in triacylglycerols (TAG) biosynthesis in oil seeds, the acylation of diacylglycerols (DAG), is catalysed by two types of enzymes: the acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). The relative contribution of these enzymes in the synthesis of TAG has not yet been defined in any plant tissue. In the presented work, microsomal preparations were obtained from sunflower and safflower seeds at different stages of development and used in DGAT and PDAT enzyme assays. The ratio between PDAT and DGAT activity differed dramatically between the two different species. DGAT activities were measured with two different acyl acceptors and assay methods using two different acyl-CoAs, and in all cases the ratio of PDAT to DGAT activity was significantly higher in safflower than sunflower. The sunflower DGAT, measured by both methods, showed significant higher activity with 18:2-CoA than with 18:1-CoA, whereas the opposite specificity was seen with the safflower enzyme. The specificities of PDAT on the other hand, were similar in both species with 18:2-phosphatidylcholine being a better acyl donor than 18:1-PC and with acyl groups at the sn-2 position utilised about fourfold the rate of the sn-1 position. No DAG:DAG transacylase activity could be detected in the microsomal preparations.

  13. Molecular phylogeny of rodents, with special emphasis on murids: evidence from nuclear gene LCAT.

    PubMed

    Robinson, M; Catzeflis, F; Briolay, J; Mouchiroud, D

    1997-12-01

    Phylogenetic relationships among 19 extant species of rodents, with special emphasis on rats, mice, and allied Muroidea, were studied using sequences of the nuclear protein-coding gene LCAT (lecithin:cholesterol acyltransferase), an enzyme of cholesterol metabolism. Analysis of 705 base pairs from the exonic regions of LCAT confirmed known groupings in and around Muroidea. Strong support was found for the families Sciuridae (squirrel and marmot) and Gliridae (dormice) and for suprafamilial taxa Muroidea and Caviomorpha (guinea pig and allies). Within Muroidea, the first branching leads to the fossorial mole rats Spalacinae and bamboo rats Rhizomyinae. The other Muroidea appear as a polytomy from which are issued Gerbillinae (gerbils), Murinae (rats and mice), Sigmodontinae (New World cricetids), Cricetinae (hamsters), and Arvicolinae (voles). Evidence from LCAT sequences agrees with that from a number of previous molecular and morphological studies, both concerning branching orders inside Muroidea and the bush-like radiation of rodent suprafamilial taxa (caviomorphs, sciurids, glirids, muroids), thus suggesting that this nuclear gene is an appropriate candidate for addressing questions of rodents relationships.

  14. 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

  15. A robust all-atom model for LCAT generated by homology modeling[S

    PubMed Central

    Segrest, Jere P.; Jones, Martin K.; Catte, Andrea; Thirumuruganandham, Saravana P.

    2015-01-01

    LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A2 (PLA2) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15–20 Å relative to PLA2. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport. PMID:25589508

  16. A robust all-atom model for LCAT generated by homology modeling.

    PubMed

    Segrest, Jere P; Jones, Martin K; Catte, Andrea; Thirumuruganandham, Saravana P

    2015-03-01

    LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A2 (PLA2) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15-20 Å relative to PLA2. ii) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii) A β-hairpin resembling a lipase lid separates S181 from solvent. iv) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specificity, and K128 and R147, whose mutations cause LCAT deficiency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport.

  17. 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.

  18. 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.

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

    PubMed

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

    2016-10-11

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

  20. 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.

  1. Metabolic tracing of monoacylglycerol acyltransferase-2 activity in vitro and in vivo.

    PubMed

    Qi, Jenson; Lang, Wensheng; Connelly, Margery A; Du, Fuyong; Liang, Yin; Caldwell, Gary W; Martin, Tonya; Hansen, Michael K; Kuo, Gee-Hong; Gaul, Michael D; Pocai, Alessandro; Lee, Seunghun

    2017-05-01

    Monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DAG) from free fatty acids (FFA) and sn-monoacylglycerol (MG), the two major hydrolysis products of dietary fat. To demonstrate MGAT2-mediated cellular activity of triglyceride (TG) synthesis, we utilized 1-oleoyl-glycerol-d5 as a substrate to trace MGAT2-driven 1-oleoyl-glycerol-d5 incorporation into TG in HEK293 cells stably expressing human MGAT2. The oleoyl-glycerol-d5 incorporated major TG species were then quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) in a 96-well format. Conventional MGAT2 target-engagement in vivo assays measure the elevation of total plasma TG by orally dosing a bolus of TG oil. We developed a novel LC/ESI/MS/MS-based fat absorption assay to assess the ability of MGAT2 inhibitors to inhibit fat absorption in CD1 mice by a meal tolerance test consisting of a mixture of liquid Boost plus(®) and 0.59 g/kg (U13)C-TG oil. The newly resynthesized plasma heavy TGs containing three (13)C in the glycerol backbone and two (U13)C-acyl-chains, which represented the digested, absorbed and resynthesized TGs, were then quantitated by LC/ESI/MS/MS. With this assay, we identified a potent MGAT2 inhibitor that blocked MGAT2-mediated activity in vitro and in vivo. The use of 1-oleoyl-glycerol-d5 and (U13)C-TG oil followed by LC/ESI/MS/MS detection of stable-isotopic labeled DAG, TG, or glycerol provides a wide range of applications to study pathophysiological regulation of the monoacylglycerol pathway and MGAT2 activity.

  2. 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...

  3. 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

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

    PubMed

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

    2013-08-01

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

  5. 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

  6. 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.

  7. 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.; ...

    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

  8. 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).

  9. 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

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

    SciTech Connect

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

    2012-08-31

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

  11. Increased mitochondrial glycerol-3-phosphate acyltransferase protein and enzyme activity in rat epididymal fat upon cessation of wheel running.

    PubMed

    Kump, David S; Laye, Matthew J; Booth, Frank W

    2006-03-01

    Triacylglycerol synthesis in rat epididymal fat overshoots sedentary levels at 10, 29, and 53 h of physical inactivity after 21 days of wheel running. The purposes of the present study were to determine 1) whether this effect is also observed after an acute bout of physical activity and 2) what enzymatic changes might contribute to this effect. We show that more than one bout of physical activity, such as that which occurs with 21 days of wheel running, is necessary for palmitic acid incorporation into triacylglyceride (triglyceride synthesis) to overshoot sedentary values, which suggests that pretranslational mechanisms may be responsible for this overshoot effect. Ten hours after 21 days of wheel running, activity of the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform, a key regulator of triacylglycerol synthesis, overshot sedentary values by 48% and remained higher than sedentary values at 29 and 53 h of reduced physical activity. The overshoot in mtGPAT1 activity was accompanied by an increase in mtGPAT protein level. Cyclic AMP response element-binding protein-binding protein level was higher in sedentary 29 h after 21 days of wheel running. AMP kinase-alpha Thr(172) phosphorylation was increased immediately after treadmill running, but decreased to sedentary values by 5 h after activity. Casein kinase-2alpha protein level and activity were unchanged. We conclude that an increase in mtGPAT protein might contribute to the overshoot in triacylglycerol synthesis.

  12. Differential effects of fenofibrate or simvastatin treatment of rats on hepatic microsomal overt and latent diacylglycerol acyltransferase activities.

    PubMed

    Waterman, Ian J; Zammit, Victor A

    2002-06-01

    Hepatic triacylglycerol secretion is elevated in insulin-resistant states. Microsomal diacylglycerol acyltransferase (DGAT) catalyzes the final reaction in the synthesis of triacylglycerol (TAG). We have previously described two DGAT activities in rat liver microsomes, one overt (cytosol-facing) and one latent (endoplasmic reticulum lumen-facing) (Owen MR, Corstorphine CG, Zammit VA: Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion. Biochem J 323:17-21, 1977). It was suggested that they are involved in the synthesis of TAG for the cytosolic droplet and VLDL lipidation, respectively. In the present study, we measured the overt and latent DGAT activities in rats fed diets containing one of two hypolipidemic drugs: fenofibrate (a peroxisome proliferator-activated receptor alpha [PPARalpha] agonist) and simvastatin (a 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitor). We found that the activities of the two DGATs could be varied independently by these treatments. Fenofibrate raised overt DGAT activity but lowered that of latent DGAT. In contrast, simvastatin markedly lowered overt DGAT activity without affecting that of latent DGAT. The increase in overt DGAT activity induced by fenofibrate could not be mimicked by feeding a diet enriched in n-3 polyunsaturated fatty acids (PUFA), which lowered overt DGAT activity but did not affect latent DGAT, suggesting that n-3 PUFA act through a mechanism independent of PPARalpha activation. The fibrate-induced increase in overt DGAT activity and the inhibition of latent DGAT may provide a mechanism through which acyl moieties are retained within the liver for oxidation through the pathways concomitantly upregulated by PPARalpha activation.

  13. Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

    PubMed Central

    Katavic, V; Reed, D W; Taylor, D C; Giblin, E M; Barton, D L; Zou, J; Mackenzie, S L; Covello, P S; Kunst, L

    1995-01-01

    In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids. PMID:7784510

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

    PubMed

    Letterie, G S

    2000-06-01

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

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

    PubMed

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

    2015-07-01

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

  16. 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...

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

    PubMed

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

    2014-07-01

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

  18. Glycerophosphate/Acylglycerophosphate Acyltransferases

    PubMed Central

    Yamashita, Atsushi; Hayashi, Yasuhiro; Matsumoto, Naoki; Nemoto-Sasaki, Yoko; Oka, Saori; Tanikawa, Takashi; Sugiura, Takayuki

    2014-01-01

    Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) and acyl-CoA: 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) are involved in the de novo synthesis of triacylglycerol (TAG) and glycerophospholipids. Many enzymes belonging to the GPAT/AGPAT family have recently been identified and their physiological or pathophysiological roles have been proposed. The roles of GPAT/AGPAT in the synthesis of TAG and obesity-related diseases were revealed through the identification of causative genes of these diseases or analyses of genetically manipulated animals. Recent studies have suggested that some isoforms of GPAT/AGPAT family enzymes are involved in the fatty acid remodeling of phospholipids. The enzymology of GPAT/AGPAT and their physiological/pathological roles in the metabolism of glycerolipids have been described and discussed in this review. PMID:25415055

  19. Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

    PubMed Central

    Tobin, M B; Fleming, M D; Skatrud, P L; Miller, J R

    1990-01-01

    The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa. Images PMID:2120195

  20. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase.

    PubMed Central

    Mikkelsen, J; Højrup, P; Rasmussen, M M; Roepstorff, P; Knudsen, J

    1985-01-01

    Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases. PMID:3922356

  1. Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3 beta-thiol as a fatty acyl acceptor.

    PubMed

    Zhou, G; Dolphin, P J

    1995-09-14

    Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3 beta-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 beta-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin/thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The Km for thiocholesterol was 203.6 microM with a Vmax of 5.3 nmol thiocholesteryl ester formed/h per microgram. The Km for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 microM with a Vmax of 8.8 nmol cholesteryl ester formed/h per microgram. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3 beta-SH group of thiocholesterol forming a cholesteryl thioester. Although the 3 beta-SH group is not as good a terminal acceptor as the 3 beta-OH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester.

  2. Allostery and Conformational Dynamics in cAMP-binding Acyltransferases*

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-06-06

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

  4. Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

    PubMed

    Thacker, Seth G; Rousset, Xavier; Esmail, Safiya; Zarzour, Abdalrahman; Jin, Xueting; Collins, Heidi L; Sampson, Maureen; Stonik, John; Demosky, Stephen; Malide, Daniela A; Freeman, Lita; Vaisman, Boris L; Kruth, Howard S; Adelman, Steven J; Remaley, Alan T

    2015-07-01

    LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

  5. Power Systems Life Cycle Analysis Tool (Power L-CAT).

    SciTech Connect

    Andruski, Joel; Drennen, Thomas E.

    2011-01-01

    The Power Systems L-CAT is a high-level dynamic model that calculates levelized production costs and tracks environmental performance for a range of electricity generation technologies: natural gas combined cycle (using either imported (LNGCC) or domestic natural gas (NGCC)), integrated gasification combined cycle (IGCC), supercritical pulverized coal (SCPC), existing pulverized coal (EXPC), nuclear, and wind. All of the fossil fuel technologies also include an option for including carbon capture and sequestration technologies (CCS). The model allows for quick sensitivity analysis on key technical and financial assumptions, such as: capital, O&M, and fuel costs; interest rates; construction time; heat rates; taxes; depreciation; and capacity factors. The fossil fuel options are based on detailed life cycle analysis reports conducted by the National Energy Technology Laboratory (NETL). For each of these technologies, NETL's detailed LCAs include consideration of five stages associated with energy production: raw material acquisition (RMA), raw material transport (RMT), energy conversion facility (ECF), product transportation and distribution (PT&D), and end user electricity consumption. The goal of the NETL studies is to compare existing and future fossil fuel technology options using a cradle-to-grave analysis. The NETL reports consider constant dollar levelized cost of delivered electricity, total plant costs, greenhouse gas emissions, criteria air pollutants, mercury (Hg) and ammonia (NH3) emissions, water withdrawal and consumption, and land use (acreage).

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

    SciTech Connect

    Lohman, Jeremy R.; Bingman, Craig A.; George N. Phillips Jr.; Shen, Ben

    2013-01-15

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

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

    PubMed

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

    2008-01-01

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

  8. 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.

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

    PubMed

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

    2004-01-01

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

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

    PubMed

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

    2013-03-01

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

  11. Molecular mechanism of reverse cholesterol transport: reaction of pre-beta-migrating high-density lipoprotein with plasma lecithin/cholesterol acyltransferase.

    PubMed

    Nakamura, Yasushi; Kotite, Leila; Gan, Yonghong; Spencer, Thomas A; Fielding, Christopher J; Fielding, Phoebe E

    2004-11-23

    A 70-75 kDa high-density lipoprotein (HDL) particle with pre-beta-electrophoretic migration (pre-beta(1)-HDL) has been identified in several studies as an early acceptor of cell-derived cholesterol. However, the further metabolism of this complex has not been determined. Here we sought to identify the mechanism by which cell-derived cholesterol was esterified and converted to mature HDL as part of reverse cholesterol transport (RCT). Human plasma selectively immunodepleted of pre-beta(1)-HDL was used to study factors regulating pre-beta(1)-HDL production. A major role for phospholipid transfer protein (PLTP) in the recycling of pre-beta(1)-HDL was identified. Cholesterol binding, esterification by lecithin/cholesterol acyltransferase (LCAT) and transfer by cholesteryl ester transfer protein (CETP) were measured using (3)H-cholesterol-labeled cell monolayers. LCAT bound to (3)H-free cholesterol (FC)-labeled pre-beta(1)-HDL generated cholesteryl esters at a rate much greater than the rest of HDL. The cholesteryl ester produced in pre-beta(1)-HDL in turn became the preferred substrate of CETP. Selective LCAT-mediated reactivity with pre-beta(1)-HDL represents a novel mechanism increasing the efficiency of RCT.

  12. The Canonical DHHC Motif Is Not Absolutely Required for the Activity of the Yeast S-acyltransferases Swf1 and Pfa4*

    PubMed Central

    González Montoro, Ayelén; Chumpen Ramirez, Sabrina; Valdez Taubas, Javier

    2015-01-01

    Protein S-acyltransferases, also known as palmitoyltransferases (PATs), are characterized by the presence of a 50-amino acid domain called the DHHC domain. Within this domain, these four amino acids constitute a highly conserved motif. It has been proposed that the palmitoylation reaction occurs through a palmitoyl-PAT covalent intermediate that involves the conserved cysteine in the DHHC motif. Mutation of this cysteine results in lack of function for several PATs, and DHHA or DHHS mutants are used regularly as catalytically inactive controls. In a genetic screen to isolate loss-of-function mutations in the yeast PAT Swf1, we isolated an allele encoding a Swf1 DHHR mutant. Overexpression of this mutant is able to partially complement a swf1Δ strain and to acylate the Swf1 substrates Tlg1, Syn8, and Snc1. Overexpression of the palmitoyltransferase Pfa4 DHHA or DHHR mutants also results in palmitoylation of its substrate Chs3. We also investigated the role of the first histidine of the DHHC motif. A Swf1 DQHC mutant is also partially active but a DQHR is not. Finally, we show that Swf1 substrates are differentially modified by both DHHR and DQHC Swf1 mutants. We propose that, in the absence of the canonical mechanism, alternative suboptimal mechanisms take place that are more dependent on the reactivity of the acceptor protein. These results also imply that caution must be exercised when proposing non-canonical roles for PATs on the basis of considering DHHC mutants as catalytically inactive and, more generally, contribute to an understanding of the mechanism of protein palmitoylation PMID:26224664

  13. Fish protein hydrolysate reduces plasma total cholesterol, increases the proportion of HDL cholesterol, and lowers acyl-CoA:cholesterol acyltransferase activity in liver of Zucker rats.

    PubMed

    Wergedahl, Hege; Liaset, Bjørn; Gudbrandsen, Oddrun Anita; Lied, Einar; Espe, Marit; Muna, Ziad; Mørk, Sverre; Berge, Rolf K

    2004-06-01

    There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of Delta 5 and Delta 6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein-fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient.

  14. Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice[S

    PubMed Central

    Thacker, Seth G.; Rousset, Xavier; Esmail, Safiya; Zarzour, Abdalrahman; Jin, Xueting; Collins, Heidi L.; Sampson, Maureen; Stonik, John; Demosky, Stephen; Malide, Daniela A.; Freeman, Lita; Vaisman, Boris L.; Kruth, Howard S.; Adelman, Steven J.; Remaley, Alan T.

    2015-01-01

    LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles. PMID:25964513

  15. 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...

  16. 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.

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

    SciTech Connect

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

    1986-05-01

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

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

    PubMed

    Rose, H G; Ellerbe, P

    1982-09-14

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

  19. CFD Analysis of Flexible Thermal Protection System Shear Configuration Testing in the LCAT Facility

    NASA Technical Reports Server (NTRS)

    Ferlemann, Paul G.

    2014-01-01

    This paper documents results of computational analysis performed after flexible thermal protection system shear configuration testing in the LCAT facility. The primary objectives were to predict the shear force on the sample and the sensitivity of all surface properties to the shape of the sample. Bumps of 0.05, 0.10,and 0.15 inches were created to approximate the shape of some fabric samples during testing. A large amount of information was extracted from the CFD solutions for comparison between runs and also current or future flight simulations.

  20. Lysophosphatidylcholine Acyltransferase 3 Is the Key Enzyme for Incorporating Arachidonic Acid into Glycerophospholipids during Adipocyte Differentiation

    PubMed Central

    Eto, Miki; Shindou, Hideo; Koeberle, Andreas; Harayama, Takeshi; Yanagida, Keisuke; Shimizu, Takao

    2012-01-01

    Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation. PMID:23208369

  1. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    PubMed

    Khayatt, Barzan I; Overmars, Lex; Siezen, Roland J; Francke, Christof

    2013-01-01

    There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

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

    PubMed Central

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

    1997-01-01

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

  3. 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 ...

  4. 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...

  5. Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

    PubMed Central

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

    2016-01-01

    Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells). PMID:27758859

  6. Inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) as hypocholesterolemic agents: synthesis and structure-activity relationships of novel series of sulfonamides, acylphosphonamides and acylphosphoramidates.

    PubMed

    Lee, H T; Roark, W H; Picard, J A; Sliskovic, D R; Roth, B D; Stanfield, R L; Hamelehle, K L; Bousley, R F; Krause, B R

    1998-02-03

    Sulfoacetic acid, phosphoramidate, and phosphoramide analogs of the ACAT inhibitors, CI-999 and CI-1011 were synthesized. The structure-activity relationships of these compounds as ACAT inhibitors are described.

  7. Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

    PubMed Central

    Lee, H J; Ryu, J M; Jung, Y H; Lee, K H; Kim, D I; Han, H J

    2016-01-01

    Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation. PMID:27010859

  8. Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity.

    PubMed

    Steeghs, Liana; Berns, Mariska; ten Hove, Jan; de Jong, Ad; Roholl, Paul; van Alphen, Loek; Tommassen, Jan; van der Ley, Peter

    2002-09-01

    A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2004-03-08

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

  11. A Novel Polyamine Acyltransferase Responsible for the Accumulation of Spermidine Conjugates in Arabidopsis Seed[W][OA

    PubMed Central

    Luo, Jie; Fuell, Christine; Parr, Adrian; Hill, Lionel; Bailey, Paul; Elliott, Katherine; Fairhurst, Shirley A.; Martin, Cathie; Michael, Anthony J.

    2009-01-01

    Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo. PMID:19168716

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

    PubMed

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

    2005-02-01

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

  13. Polyketide Proofreading by an Acyltransferase-like Enzyme

    PubMed Central

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

    2012-01-01

    SUMMARY Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for polyketide titer improvement and biosynthetic investigations. PMID:22444588

  14. The bushlike radiation of muroid rodents is exemplified by the molecular phylogeny of the LCAT nuclear gene.

    PubMed

    Michaux, J; Catzeflis, F

    2000-11-01

    Phylogenetic relationships among 40 extant species of rodents, with an emphasis on the taxonomic sampling of Muridae and Dipodidae, were studied using sequences of the nuclear protein-coding gene LCAT (lecithin cholesterol acyl transferase). Analysis of 804 bp from the exonic regions of LCAT confirmed many traditional groupings in and around Muridae. A strong support was found for the families Muridae (represented by 29 species) and Dipodidae (5 species). Compared with Sciuridae, Gliridae, and Caviomorpha, the Dipodidae family appeared the closest relative of Muridae, confirming the suprafamilial Myodonta concept. Within the speciose family Muridae, the first branching leads to the fossorial Spalacinae and semifossorial Rhyzomyinae. The remaining components of Muridae appear as a polytomy from which are issued Sigmodontinae, Calomyscinae, Arvicolinae, Cricetinae, Mystromyinae, Nesomyinae, and some Dendromurinae (Steatomys and Dendromus). This phylogeny is interpreted as the result of a bushlike radiation at the end of the early Miocene, leading to emergence of most living subfamilies. The separation between three additional taxa, Murinae, Gerbillinae, and "Acomyinae" (which comprises the genera Acomys, Deomys, Uranomys, and Lophuromys), has occurred more recently from a common ancestor issued from the main basal radiation. As previously shown by other molecular studies, the vlei rats, Otomyinae, are nested within Old World Murinae. In the same way, the zokors, Myospalacinae, appear strongly nested within the hamsters, Cricetinae. Finally, we propose a sister group relationship between Malagasy Nesomyinae and south African Mystromyinae.

  15. 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

  16. Action of lecithin:cholesterol acyltransferase on model lipoproteins. Preparation and characterization of model nascent high density lipoprotein.

    PubMed

    Pownall, H J; Van Winkle, W B; Pao, Q; Rohde, M; Gotto, A M

    1982-12-13

    Apolipoprotein A-I, the major protein of human plasma high density lipoprotein, is the primary activator of plasma lecithin:cholesterol acyltransferase. In vitro, the association of apolipoprotein A-I with physiological phosphatidylcholines can be catalyzed by mixing the protein and lipid with sodium cholate, which is removed by chromatography. The apolipoprotein A-I/phospholipid complex has the physical properties of an HDL, and when cholesterol is present the complex is a highly reactive substrate in the lecithin:cholesterol acyltransferase-catalyzed reaction. The relative reactivity of this complex compared with a number of other lipid-protein complexes is presented and discussed.

  17. Effects of F-1394, an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on ACAT activity in HepG2 cells and on hepatic secretion of lipids in Triton WR-1339-induced hyperlipidemic rats: possible role of hepatic ACAT in very low density lipoprotein secretion.

    PubMed

    Aragane, K; Kusunoki, J; Kitamine, T; Yamaura, T; Ohnishi, H

    1998-03-01

    We examined the inhibitory potency of F-1394 ((1S,2S)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]cyclohexane -1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on ACAT activity and its hypolipidemic effect. F-1394 inhibited whole-cell ACAT activity in HepG2 cells with an IC50 value of 42 nM. The potency of F-1394 was greater than that of the five other ACAT inhibitors tested (YM-17E, CI-976, 57-118, CL-277,082 and DL-melinamide). In rats made hyperlipidemic by Triton WR-1339, F-1394 caused a reduction in the hepatic secretion rate of cholesterol. These data suggest that inhibition of hepatic ACAT activity helps to reduce very low density lipoprotein secretion from the liver into the circulation.

  18. Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil1[OPEN

    PubMed Central

    Stymne, Sten

    2017-01-01

    Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C. sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine. PMID:28235891

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

    PubMed

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

    2015-01-01

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

  20. Does triacylglycerol biosynthesis require diacylglycerol acyltransferase (DAGAT)?

    PubMed

    Fraser, T; Waters, A; Chatrattanakunchai, S; Stobart, K

    2000-12-01

    Microsomal membrane preparations from the developing seeds of sunflower (Helianthus annuus L.) catalyse the conversion of sn-glycerol-3-phosphate and acyl-CoA to triacylglycerol via phosphatidic acid and diacylglycerol. The formation of diacylglycerol from phosphatidic acid was Mg2+ dependent and in the presence of EDTA phosphatidic acid accumulated. This property was used to generate large quantities of endogenous radioactive phosphatidic acid in the membranes. On addition of Mg2+ the phosphatidic acid was used in triacylglycerol formation. Acyl-CoA had little effect on the label which accumulated in triacylglycerol from phosphatidic acid. Diacylglycerol acyltransferase, therefore, may not play a major role in oil formation as originally envisaged and other enzymes, including diacylglycerol:diacylglycerol transacylase [Stobart, Mancha, Lenman, Dahlqvist and Stymne (1997) Planta 203, 58-66] may have important biosynthetic functions.

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

    PubMed

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

    2012-10-01

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

  2. Dietary fat modulates serum paraoxonase 1 activity in rats.

    PubMed

    Kudchodkar, B J; Lacko, A G; Dory, L; Fungwe, T V

    2000-10-01

    We examined the effects of dietary fats with specific fatty acid compositions, on serum paraoxonase (PON1) activity in rats. Male adult Sprague-Dawley rats were divided randomly into four dietary groups. One group received the control diet [AIN 93M with soybean oil (5 g/100 g diet)], whereas the remaining three groups received the modified control diet supplemented with (15 g/100 g diet) triolein, tripalmitin or fish oil, respectively. After 20 d, blood was obtained after overnight food deprivation and PON1 activity was determined. Serum lipids and lipid components of lipoproteins were also determined. Serum PON1 activity [micromol/(L.min)] was significantly (P: < 0.05) higher in triolein (98 +/- 6) and lower in fish oil (41 +/- 4), compared with tripalmitin-fed rats (63 +/- 11). Serum PON1 activity in tripalmitin-fed rats was comparable to that of controls (67 +/- 9). Serum PON1 activity correlated significantly with serum lecithin:cholesterol acyltransferase (LCAT) activity (r = 0.77, P: < 0.001) and was transported in blood principally in association with the denser subfraction of HDL, very high density lipoprotein (VHDL; d > 1.15 kg/L). Serum PON1 activity correlated strongly with serum lipids as well as lipids of VLDL, HDL and its subfractions. Multiple linear regression analysis, however, showed a significant relationship of serum PON1 activity, principally with the phospholipids of VHDL (r = 0.47, P: < 0.002). These data suggest that the modulation of serum PON1 activity by dietary fat may be mediated via the effect of the specific fatty acids on the synthesis and secretion of VHDL, the subfraction of HDL that transports the majority of PON1 in the blood.

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

    PubMed

    Dunn, Briana J; Khosla, Chaitan

    2013-08-06

    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.

  4. 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.

  5. 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

  6. 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.

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

    PubMed

    Kalscheuer, Rainer; Steinbüchel, Alexander

    2003-03-07

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

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

    PubMed Central

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

    2012-01-01

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

  9. Polyketide proofreading by an acyltransferase-like enzyme.

    PubMed

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

    2012-03-23

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

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

    PubMed

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

    2009-01-01

    The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p < 10(-8)) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

  11. A calcium-dependent acyltransferase that produces N-acyl phosphatidylethanolamines

    PubMed Central

    Ogura, Yuji; Parsons, William H.; Kamat, Siddhesh S.; Cravatt, Benjamin F.

    2016-01-01

    More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells. PMID:27399000

  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. Cloning and Functional Characterization of a Phospholipid:Diacylglycerol Acyltransferase from Arabidopsis1

    PubMed Central

    Ståhl, Ulf; Carlsson, Anders S.; Lenman, Marit; Dahlqvist, Anders; Huang, Bangquan; Banaś, Walentyna; Banaś, Antoni; Stymne, Sten

    2004-01-01

    A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487–6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type. PMID:15247387

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

    PubMed

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

    2010-05-15

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

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

    PubMed

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

    2012-12-15

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors.

    PubMed

    Qi, Jenson; Masucci, John A; Lang, Wensheng; Connelly, Margery A; Caldwell, Gary W; Petrounia, Ioanna; Kirkpatrick, Jennifer; Barnakov, Alexander N; Struble, Geoffrey; Miller, Robyn; Dzordzorine, Keli; Kuo, Gee-Hong; Gaul, Michael; Pocai, Alessandro; Lee, Seunghun

    2017-04-01

    Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

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

    PubMed

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

    2006-06-06

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

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

    PubMed

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

    2009-10-02

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

    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.

    2015-01-01

    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.—Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification. PMID:26220175

  6. 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.

  7. 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

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

    PubMed

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

    2009-05-08

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

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

    PubMed

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

    2012-10-01

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

  10. Function and Localization of the Arabidopsis thaliana Diacylglycerol Acyltransferase DGAT2 Expressed in Yeast

    PubMed Central

    Aymé, Laure; Baud, Sébastien; Dubreucq, Bertrand; Joffre, Florent; Chardot, Thierry

    2014-01-01

    Diacylglycerol acyltransferases (DGATs) catalyze the final and only committed step of triacylglycerol synthesis. DGAT activity is rate limiting for triacylglycerol accumulation in mammals, plants and microbes. DGATs belong to three different evolutionary classes. In Arabidopsis thaliana, DGAT1, encoded by At2g19450, is the major DGAT enzyme involved in triacylglycerol accumulation in seeds. Until recently, the function of DGAT2 (At3g51520) has remained elusive. Previous attempts to characterize its enzymatic function by heterologous expression in yeast were unsuccessful. In the present report we demonstrate that expression of a codon-optimized version of the DGAT2 gene is able to restore neutral lipid accumulation in the Saccharomyces cerevisiae mutant strain (H1246), which is defective in triacylglycerol biosynthesis. Heterologous expression of codon-optimized DGAT2 and DGAT1 induced the biogenesis of subcellular lipid droplets containing triacylglycerols and squalene. Both DGAT proteins were found to be associated with these lipid droplets. The fatty acid composition was affected by the nature of the acyltransferase expressed. DGAT2 preferentially incorporated C16:1 fatty acids whereas DGAT1 displayed preference for C16:0, strongly suggesting that these enzymes have contrasting substrate specificities. PMID:24663078

  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. 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.

  13. Four Acyltransferases Uniquely Contribute to Phospholipid Heterogeneity in Saccharomyces cerevisiae

    PubMed Central

    Oelkers, Peter; Pokhrel, Keshav

    2016-01-01

    Diverse acyl-CoA species and acyltransferase isoenzymes are components of a complex system that synthesizes glycerophospholipids and triacylglycerols. Saccharomyces cerevisiae has four main acyl-CoA species, two main glycerol-3-phosphate 1-O-acyltransferases (Gat1p, Gat2p), and two main 1-acylglycerol-3-phosphate O-acyltransferases (Lpt1p, Slc1p). The in vivo contribution of these isoenzymes to phospholipid heterogeneity was determined using haploids with compound mutations: gat1Δlpt1Δ, gat2Δlpt1Δ, gat1Δslc1Δ, and gat2Δslc1Δ. All mutations mildly reduced [3H]palmitic acid incorporation into phospholipids relative to triacylglycerol. Electrospray ionization tandem mass spectrometry identified few differences from wild type in gat1Δlpt1Δ, dramatic differences in gat2Δslc1Δ, and intermediate changes in gat2Δlpt1Δ and gat1Δslc1Δ. Yeast expressing Gat1p and Lpt1p had phospholipids enriched with acyl chains that were unsaturated, 18 carbons long, and paired for length. These alterations prevented growth at 18.5°C and in 10% ethanol. Therefore, Gat2p and Slc1p dictate phospholipid acyl chain composition in rich media at 30°C. Slc1p selectively pairs acyl chains of different lengths. PMID:27920551

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

    Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. PMID:26395841

  16. 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.

  17. Apolipoprotein AI tertiary structures determine stability and phospholipid-binding activity of discoidal high-density lipoprotein particles of different sizes

    SciTech Connect

    Chen, Bin; Ren, Xuefeng; Neville, Tracey; Jerome, W. Gray; Hoyt, David W.; Sparks, Daniel L.; Ren, Gang; Wang, Jianjun

    2009-05-18

    Human high-density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140-240 kDa, disk-shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase (LCAT). One of the major problems for high-resolution structural studies of discoidal HDL is the difficulty in obtaining pure and, foremost, homogenous sample. We demonstrate here that the commonly used cholate dialysis method for discoidal HDL preparation usually contains 5-10% lipid-poor apoAI that significantly interferes with the high-resolution structural analysis of discoidal HDL using biophysical methods. Using an ultracentrifugation method, we quickly removed lipid-poor apoAI. We also purified discoidal reconstituted HDL (rHDL) into two pure discoidal HDL species of different sizes that are amendable for high-resolution structural studies. A small rHDL has a diameter of 7.6 nm, and a large rHDL has a diameter of 9.8 nm. We show that these two different sizes of discoidal HDL particles display different stability and phospholipid-binding activity. Interestingly, these property/functional differences are independent from the apoAI -helical secondary structure, but are determined by the tertiary structural difference of apoAI on different discoidal rHDL particles, as evidenced by two-dimensional NMR and negative stain electron microscopy data. Our result further provides the first high-resolution NMR data, demonstrating a promise of structural determination of discoidal HDL at atomic resolution using a combination of NMR and other biophysical techniques.

  18. Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis.

    PubMed

    Lung, Shiu-Cheung; Weselake, Randall J

    2006-12-01

    Many plants deposit TAG in seeds and fruits as the major form of storage lipid. TAG production is of tremendous socioeconomic value in food, nutraceutical, and industrial applications, and thus numerous conventional and molecular genetic strategies have been explored in attempts to increase TAG content and modify the FA composition of plant seed oils. Much research has focused on the acyl-CoA-dependent reaction catalyzed by diacylglycerol acyltransferase (DGAT), which is an integral endoplasmic reticulum protein and has also been shown to be present in oil bodies and plastids. DGAT enzymes exhibit diverse biochemical properties among different plant species, many of which are summarized here. In addition to catalyzing a critical step in TAG biosynthesis, there is evidence that DGAT has roles in lipid metabolism associated with germination and leaf senescence. TAG can also be formed in plants via two different acyl-CoA-independent pathways, catalyzed by phospholipid: diacylglycerol acyltransferase and diacylglycerol transacylase. The current understanding of the terminal step in TAG formation in plants and the development of molecular genetic approaches aimed at altering TAG yield and FA composition of TAG are discussed.

  19. Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

    PubMed Central

    Röttig, Annika; Strittmatter, Carl Simon; Schauer, Jennifer; Hiessl, Sebastian; Daniel, Rolf

    2016-01-01

    ABSTRACT Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli. The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCE A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected

  20. Glycerolipid biosynthesis in rat adipose tissue. I. Properties and distribution of glycerophosphate acyltransferase and effect of divalent cations on neutral lipid formation.

    PubMed

    Jamdar, S C; Fallon, H J

    1973-09-01

    A sensitive radioactive assay of acyl CoA:sn-glycerol-3-phosphate-O-acyltransferase (EC 2.3.1.15) was developed to study the properties and subcellular distribution of this enzyme in rat epididymal adipose tissue. The esterification of sn-glycerol-3-phosphate was measured in the presence of palmitoyl CoA or palmitate, ATP, CoA, and Mg(2+) at pH 7.5. The presence of glycerophosphate acyltransferase was detected in both mitochondria and microsomes. The product of this reaction was identified as phosphatidate by thin-layer chromatography and dual isotope incorporation studies. Several divalent cations reduced the activity of this enzyme. Although Mg(2+) was not required for the activity of glycerophosphate acyltransferase, its addition to the incubation mixture resulted in an increased formation of neutral lipids at the expense of phosphatidate. This result is explained by an activation of microsomal phosphatidate phosphatase (EC 3.1.3.4). The effect of Mg(2+) was completely abolished by Ni(2+), Co(2+), Mn(2+), and Zn(2+). These studies suggest that the balance between Mg(2+) and several other divalent ions may be important in the regulation of neutral lipid synthesis in adipose tissue.

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

    PubMed

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

    2010-09-01

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

  2. Molecular cloning and biochemical characterization of Candida albicans acyl-CoA:sterol acyltransferase, a potential target of antifungal agents.

    PubMed

    Kim, Ki-Young; Shin, Yu-Kyong; Park, Jong-Chul; Kim, Jung-Ho; Yang, Hongyuan; Han, Dong-Min; Paik, Young-Ki

    2004-07-02

    To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.

  3. 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.

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

    PubMed

    Sternberg, Robin M; LeBlanc, Gerald A

    2006-06-30

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

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

    PubMed

    Frankel, Brenda A; Blanchard, John S

    2008-09-15

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

  6. 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

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

    PubMed Central

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

    2012-01-01

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

  8. 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

  9. 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

  10. 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

    2016-03-11

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

  11. Automation of PRM-dependent D3-Leu tracer enrichment in HDL to study the metabolism of apoA-I, LCAT and other apolipoproteins.

    PubMed

    Lee, Lang Ho; Andraski, Allison B; Pieper, Brett; Higashi, Hideyuki; Sacks, Frank M; Aikawa, Masanori; Singh, Sasha A

    2017-01-01

    We developed an automated quantification workflow for PRM-enabled detection of D3-Leu labeled apoA-I in high-density lipoprotein (HDL) isolated from humans. Subjects received a bolus injection of D3-Leu and blood was drawn at eight time points over three days. HDL was isolated and separated into six size fractions for subsequent proteolysis and PRM analysis for the detection of D3-Leu signal from ∼0.03 to 0.6% enrichment. We implemented an intensity-based quantification approach that takes advantage of high-resolution/accurate mass PRM scans to identify the D3-Leu 2HM3 ion from non-specific peaks. Our workflow includes five modules for extracting the targeted PRM peak intensities (XPIs): Peak centroiding, noise removal, fragment ion matching using Δm/z windows, nine intensity quantification options, and validation and visualization outputs. We optimized the XPI workflow using in vitro synthesized and clinical samples of D0/D3-Leu labeled apoA-I. Three subjects' apoA-I enrichment curves in six HDL size fractions, and LCAT, apoA-II and apoE from two size fractions were generated within a few hours. Our PRM strategy and automated quantification workflow will expedite the turnaround of HDL apoA-I metabolism data in clinical studies that aim to understand and treat the mechanisms behind dyslipidemia.

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

    PubMed

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

    2003-05-01

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

  13. 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.

  14. 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

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

    PubMed

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

    2016-06-01

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

  16. Sterol O-Acyltransferase 2 Contributes to the Yolk Cholesterol Trafficking during Zebrafish Embryogenesis

    PubMed Central

    Lee, Yen-Hua; HuangFu, Wei-Chun

    2016-01-01

    To elucidate whether Sterol O-acyltransferase (Soat) mediates the absorption and transportation of yolk lipids to the developing embryo, zebrafish soat1 and soat2 were cloned and studied. In the adult zebrafish, soat1 was detected ubiquitously while soat2 mRNA was detected specifically in the liver, intestine, brain and testis. Whole mount in situ hybridization demonstrated that both soat1 and soat2 expressed in the yolk syncytial layer, hatching gland and developing cardiovascular as well as digestive systems, suggesting that Soats may play important roles in the lipid trafficking and utilization during embryonic development. The enzymatic activity of zebrafish Soat2 was confirmed by Oil Red O staining in the HEK293 cells overexpressing this gene, and could be quenched by Soat2 inhibitor Pyripyropene A (PPPA). The zebrafish embryos injected with PPPA or morpholino oligo against soat2 in the yolk showed significantly larger yolk when compared with wild-type embryos, especially at 72 hpf, indicating a slower rate of yolk consumption. Our result indicated that zebrafish Soat2 is catalytically active in synthesizing cholesteryl esters and contributes to the yolk cholesterol trafficking during zebrafish embryogenesis. PMID:27936201

  17. 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.

  18. Inversion of Extender Unit Selectivity in the Erythromycin Polyketide Synthase by Acyltransferase Domain Engineering.

    PubMed

    Koryakina, Irina; Kasey, Christian; McArthur, John B; Lowell, Andrew N; Chemler, Joseph A; Li, Shasha; Hansen, Douglas A; Sherman, David H; Williams, Gavin J

    2017-01-20

    Acyltransferase (AT) domains of polyketide synthases (PKSs) select extender units for incorporation into polyketides and dictate large portions of the structures of clinically relevant natural products. Accordingly, there is significant interest in engineering the substrate specificity of PKS ATs in order to site-selectively manipulate polyketide structure. However, previous attempts to engineer ATs have yielded mutant PKSs with relaxed extender unit specificity, rather than an inversion of selectivity from one substrate to another. Here, by directly screening the extender unit selectivity of mutants from active site saturation libraries of an AT from the prototypical PKS, 6-deoxyerythronolide B synthase, a set of single amino acid substitutions was discovered that dramatically impact the selectivity of the PKS with only modest reductions of product yields. One particular substitution (Tyr189Arg) inverted the selectivity of the wild-type PKS from its natural substrate toward a non-natural alkynyl-modified extender unit while maintaining more than twice the activity of the wild-type PKS with its natural substrate. The strategy and mutations described herein form a platform for combinatorial biosynthesis of site-selectively modified polyketide analogues that are modified with non-natural and non-native chemical functionality.

  19. Diversity and evolution of plant diacylglycerol acyltransferase (DGATs) unveiled by phylogenetic, gene structure and expression analyses

    PubMed Central

    Turchetto-Zolet, Andreia Carina; Christoff, Ana Paula; Kulcheski, Franceli Rodrigues; Loss-Morais, Guilherme; Margis, Rogerio; Margis-Pinheiro, Marcia

    2016-01-01

    Abstract Since the first diacylglycerol acyltransferase (DGAT) gene was characterized in plants, a number of studies have focused on understanding the role of DGAT activity in plant triacylglycerol (TAG) biosynthesis. DGAT enzyme is essential in controlling TAGs synthesis and is encoded by different genes. DGAT1 and DGAT2 are the two major types of DGATs and have been well characterized in many plants. On the other hand, the DGAT3 and WS/DGAT have received less attention. In this study, we present the first general view of the presence of putative DGAT3 and WS/DGAT in several plant species and report on the diversity and evolution of these genes and its relationships with the two main DGAT genes (DGAT1 and DGAT2). According to our analyses DGAT1, DGAT2, DGAT3 and WS/DGAT are very divergent genes and may have distinct origin in plants. They also present divergent expression patterns in different organs and tissues. The maintenance of several types of genes encoding DGAT enzymes in plants demonstrates the importance of DGAT activity for TAG biosynthesis. Evolutionary history studies of DGATs coupled with their expression patterns help us to decipher their functional role in plants, helping to drive future biotechnological studies. PMID:27706370

  20. 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.

  1. 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

  2. The acyltransferase LYCAT controls specific phosphoinositides and related membrane traffic

    PubMed Central

    Bone, Leslie N.; Dayam, Roya M.; Lee, Minhyoung; Kono, Nozomu; Fairn, Gregory D.; Arai, Hiroyuki; Botelho, Roberto J.; Antonescu, Costin N.

    2017-01-01

    Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIP-dependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P2. Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology. PMID:28035047

  3. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells.

    PubMed

    Rodgers, Ursula R; Lanyon-Hogg, Thomas; Masumoto, Naoko; Ritzefeld, Markus; Burke, Rosemary; Blagg, Julian; Magee, Anthony I; Tate, Edward W

    2016-12-16

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function.

  4. Increased penicillin production in Penicillium chrysogenum production strains via balanced overexpression of isopenicillin N acyltransferase.

    PubMed

    Weber, Stefan S; Polli, Fabiola; Boer, Rémon; Bovenberg, Roel A L; Driessen, Arnold J M

    2012-10-01

    Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

  5. 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.

  6. 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

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

    PubMed

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

    2013-01-18

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

  8. Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

    PubMed Central

    Rossin, A; Durivault, J; Chakhtoura-Feghali, T; Lounnas, N; Gagnoux-Palacios, L; Hueber, A-O

    2015-01-01

    The death receptor Fas undergoes a variety of post-translational modifications including S-palmitoylation. This protein acylation has been reported essential for an optimal cell death signaling by allowing both a proper Fas localization in cholesterol and sphingolipid-enriched membrane nanodomains, as well as Fas high-molecular weight complexes. In human, S-palmitoylation is controlled by 23 members of the DHHC family through their palmitoyl acyltransferase activity. In order to better understand the role of this post-translational modification in the regulation of the Fas-mediated apoptosis pathway, we performed a screen that allowed the identification of DHHC7 as a Fas-palmitoylating enzyme. Indeed, modifying DHHC7 expression by specific silencing or overexpression, respectively, reduces or enhances Fas palmitoylation and DHHC7 co-immunoprecipitates with Fas. At a functional level, DHHC7-mediated palmitoylation of Fas allows a proper Fas expression level by preventing its degradation through the lysosomes. Indeed, the decrease of Fas expression obtained upon loss of Fas palmitoylation can be restored by inhibiting the lysosomal degradation pathway. We describe the modification of Fas by palmitoylation as a novel mechanism for the regulation of Fas expression through its ability to circumvent its degradation by lysosomal proteolysis. PMID:25301068

  9. Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

    PubMed Central

    Weber, Stefan S.; Polli, Fabiola; Boer, Rémon; Bovenberg, Roel A. L.

    2012-01-01

    Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme. PMID:22865068

  10. 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.

  11. Structure and mechanism of the trans-acting acyltransferase from the disorazole synthase.

    PubMed

    Wong, Fong T; Jin, Xi; Mathews, Irimpan I; Cane, David E; Khosla, Chaitan

    2011-08-02

    The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the "AT-less" disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser → Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.

  12. Characterization of Hedgehog Acyltransferase Inhibitors Identifies a Small Molecule Probe for Hedgehog Signaling by Cancer Cells

    PubMed Central

    2016-01-01

    The Sonic Hedgehog (Shh) signaling pathway plays a critical role during embryonic development and cancer progression. N-terminal palmitoylation of Shh by Hedgehog acyltransferase (Hhat) is essential for efficient signaling, raising interest in Hhat as a novel drug target. A recently identified series of dihydrothienopyridines has been proposed to function via this mode of action; however, the lead compound in this series (RUSKI-43) was subsequently shown to possess cytotoxic activity unrelated to canonical Shh signaling. To identify a selective chemical probe for cellular studies, we profiled three RUSKI compounds in orthogonal cell-based assays. We found that RUSKI-43 exhibits off-target cytotoxicity, masking its effect on Hhat-dependent signaling, hence results obtained with this compound in cells should be treated with caution. In contrast, RUSKI-201 showed no off-target cytotoxicity, and quantitative whole-proteome palmitoylation profiling with a bioorthogonal alkyne-palmitate reporter demonstrated specific inhibition of Hhat in cells. RUSKI-201 is the first selective Hhat chemical probe in cells and should be used in future studies of Hhat catalytic function. PMID:27779865

  13. 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

  14. 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

  15. LPT1 encodes a membrane-bound O-acyltransferase involved in the acylation of lysophospholipids in the yeast Saccharomyces cerevisiae.

    PubMed

    Tamaki, Hisanori; Shimada, Atsushi; Ito, Yoshihiro; Ohya, Mihoko; Takase, Juri; Miyashita, Masahiro; Miyagawa, Hisashi; Nozaki, Hiroyuki; Nakayama, Reiko; Kumagai, Hidehiko

    2007-11-23

    Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

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

    PubMed

    Kojima, Masayasu; Hamamoto, Akie; Sato, Takahiro

    2016-10-01

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

  17. Reaction of discoidal complexes of apolipoprotein A-I and various phosphatidylcholines with lecithin cholesterol acyltransferase. Interfacial effects.

    PubMed

    Jonas, A; Zorich, N L; Kézdy, K E; Trick, W E

    1987-03-25

    Complexes of phospholipids-apolipoprotein A-I-cholesterol, containing various bulk phosphatidylcholines or a matrix of the ether analog of 1-palmitoyl 2-oleoyl phosphatidylcholine including test phosphatidylcholines were used as substrates for human lecithin-cholesterol acyltransferase. The enzymatic reaction rates for both series of complexes were determined as a function of temperature, particle concentration, neutral salt concentration, and the type of anion present in solution. The kinetic results support the hypothesis that phospholipids, in discoidal complexes, modulate the reaction rates by molecular effects at the active site, but also by interfacial effects on the interaction of the enzyme with the particles. The relevant interfacial parameters are the lipid packing at the interface and the structure of apolipoprotein A-I.

  18. Characterization of mitochondrial glycerol-3-phosphate acyltransferase in notothenioid fishes.

    PubMed

    Keenan, Kelly A; Grove, Theresa J; Oldham, Corey A; O'Brien, Kristin M

    2017-02-01

    Hearts of Antarctic icefishes (suborder Notothenioidei, family Channichthyidae) have higher densities of mitochondria, and mitochondria have higher densities of phospholipids, compared to red-blooded notothenioids. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step in glycerolipid biosynthesis. There are four isoforms of GPAT in vertebrates; GPAT1 and GPAT2 are localized to the outer mitochondrial membrane, whereas GPAT3 and GPAT4 are localized to the endoplasmic reticulum membrane. We hypothesized that transcript levels of GPAT1 and/or GPAT2 would mirror densities of mitochondrial phospholipids and be higher in the icefish Chaenocephalus aceratus compared to the red-blooded species Notothenia coriiceps. Transcript levels of GPAT1 were quantified in heart ventricles and liver using qRT-PCR. Additionally, GPAT1 cDNA was sequenced in the Antarctic notothenioids, C. aceratus and N. coriiceps, and in the sub-Antarctic notothenioid, Eleginops maclovinus, to identify amino acid substitutions that may maintain GPAT1 function at cold temperature. Transcript levels of GPAT1 were higher in liver compared to heart ventricles but were not significantly different between the two species. In contrast, transcripts of GPAT2 were only detected in ventricle where they were 6.6-fold higher in C. aceratus compared to N. coriiceps. These data suggest GPAT1 may be more important for synthesizing triacylglycerol, whereas GPAT2 may regulate synthesis of phospholipids. GPAT1 amino acid sequences are highly conserved among the three notothenioids with 97.9-98.7% identity. Four amino acid substitutions within the cytosolic region of Antarctic notothenioid GPAT1 may maintain conformational changes necessary for binding and catalysis at cold temperature.

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

    PubMed

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

    2011-09-01

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

  20. 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.

  1. Identification and localization of a lipase-like acyltransferase in phenylpropanoid metabolism of tomato (Solanum lycopersicum).

    PubMed

    Teutschbein, Jenny; Gross, Wiltrud; Nimtz, Manfred; Milkowski, Carsten; Hause, Bettina; Strack, Dieter

    2010-12-03

    We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.

  2. Silencing an N-Acyltransferase-Like Involved in Lignin Biosynthesis in Nicotiana attenuata Dramatically Alters Herbivory-Induced Phenolamide Metabolism

    PubMed Central

    Onkokesung, Nawaporn; Galis, Ivan; Baldwin, Ian T.

    2013-01-01

    In a transcriptomic screen of Manduca sexta-induced N-acyltransferases in leaves of Nicotiana attenuata, we identified an N-acyltransferase gene sharing a high similarity with the tobacco lignin-biosynthetic hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) gene whose expression is controlled by MYB8, a transcription factor that regulates the production of phenylpropanoid polyamine conjugates (phenolamides, PAs). To evaluate the involvement of this HCT-like gene in lignin production as well as the resulting crosstalk with PA metabolism during insect herbivory, we transiently silenced (by VIGs) the expression of this gene and performed non-targeted (UHPLC-ESI/TOF-MS) metabolomics analyses. In agreement with a conserved function of N. attenuata HCT-like in lignin biogenesis, HCT-silenced plants developed weak, soft stems with greatly reduced lignin contents. Metabolic profiling demonstrated large shifts (up to 12% deregulation in total extracted ions in insect-attacked leaves) due to a large diversion of activated coumaric acid units into the production of developmentally and herbivory-induced coumaroyl-containing PAs (N′,N′′-dicoumaroylspermidine, N′,N′′-coumaroylputrescine, etc) and to minor increases in the most abundant free phenolics (chlorogenic and cryptochlorogenic acids), all without altering the production of well characterized herbivory-responsive caffeoyl- and feruloyl-based putrescine and spermidine PAs. These data are consistent with a strong metabolic tension, exacerbated during herbivory, over the allocation of coumaroyl-CoA units among lignin and unusual coumaroyl-containing PAs, and rule out a role for HCT-LIKE in tuning the herbivory-induced accumulation of other PAs. Additionally, these results are consistent with a role for lignification as an induced anti-herbivore defense. PMID:23704878

  3. 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...

  4. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. 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...

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

    PubMed

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

    2013-05-01

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

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

    PubMed

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

    1998-10-09

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

  9. 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.

  10. Overexpression of Peanut Diacylglycerol Acyltransferase 2 in Escherichia coli

    PubMed Central

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

    2013-01-01

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

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  12. [Acylation specificity of midecamycin 3-O-acyltransferase within Streptomyces spiramyceticus F21].

    PubMed

    Ma, Chunyan; Wu, Linzhuan; Dai, Jianlu; Zhou, Hongxia; Li, Jingyan; Sun, Xiaochun; Zhang, Kan; Xia, Huanzhang; Wang, Yiguang

    2008-12-01

    Spiramycin and midecamycin are 16-membered macrolide antibiotics with very similar chemical structures. Spiramycin has three components, namely spiramycin I, II and III. Spiramycin II and III are, respectively, the O-acetyl and propionyl derivatives at C3-hydroxyl group of spiramycin I. Midecamycin has four components, and the C3-hydroxyl group of midecamycin is all O-propionylated. The enzyme adding acyl group(s) at the C3-hydroxyl group during the biosynthesis of spiramycin and midecamycin is 3-O-acyltransferase. The 3-O-acyltransferases for spiramycin and midecamycin are also very similar, and presume to function when exchanged. To explore whether the 3-O-acyltransferase for midecamycin biosynthesis hold still the character of selective and efficient propionylation for spiramycin I at its C3-hydroxyl group, we inserted mdmB, the 3-O-acyltransferase gene from Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis, into a mutant strain of S. spiramyceticus F21, in which the 3-O-acyltransferase gene for spiramycin biosynthesis, sspA, was deleted; and the mdmB was integrated exactly into the chromosomal site where the sspA was deleted. We name this "hybrid" strain as SP-mdmB. HPLC analysis of the spiramycin produced by SP-mdmB showed that spiramycin I was still the major component, although the relative proportions of both spiramycin II and III increased significantly. We thus conclude that MdmB from Streptomyces mycarofaciens ATCC 21454 for midecamyicn biosynthesis do not hold the character of selective and efficient propionylation for spiramycin I within S. spiramyceticus F21, and this character is possibly limited in Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis.

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

    PubMed

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

    2007-02-01

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

  14. Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase.

    PubMed

    Kim, Ji-Hyun; Lee, Hyo-Jung; Jeong, Soo-Jin; Lee, Min-Ho; Kim, Sung-Hoon

    2012-09-01

    Hyperlipidemia is an important factor to induce metabolic syndrome such as obesity, diabetes and cardiovascular diseases. Recently, some antihyperlipidemic agents from herbal medicines have been in the spotlight in the medical science field. Thus, the present study evaluated the antihyperlipidemic activities of the essential oil from the leaves of Pinus koraiensis SIEB (EOPK) that has been used as a folk remedy for heart disease. The reverse transcription polymerase chain reaction (RT-PCR) revealed that EOPK up-regulated low density lipoprotein receptor (LDLR) at the mRNA level as well as negatively suppressed the expression of sterol regulatory element-binding protein (SREBP)-1c, SREBP-2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) involved in lipid metabolism in HepG2 cells. Also, western blotting showed that EOPK activated LDLR and attenuated the expression of FAS at the protein level in the cells. Consistently, EOPK significantly inhibited the level of human acylcoenzyme A: cholesterol acyltransferase (hACAT)1 and 2 and reduced the low-density lipoprotein (LDL) oxidation activity. Furthermore, chromatography-mass spectrometry (GC-MS) analysis showed that EOPK, an essential oil mixture, contained camphene (21.11%), d-limonene (21.01%), α-pinene (16.74%) and borneol (11.52%). Overall, the findings suggest that EOPK can be a potent pharmaceutical agent for the prevention and treatment of hyperlipidemia.

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

    PubMed Central

    Ruan, X; Pereda, A; Stassi, D L; Zeidner, D; Summers, R G; Jackson, M; Shivakumar, A; Kakavas, S; Staver, M J; Donadio, S; Katz, L

    1997-01-01

    The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds. PMID:9335291

  18. 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.

  19. Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor degeneration in rd11 mice

    PubMed Central

    Friedman, James S.; Chang, Bo; Krauth, Daniel S.; Lopez, Irma; Waseem, Naushin H.; Hurd, Ron E.; Feathers, Kecia L.; Branham, Kari E.; Shaw, Manessa; Thomas, George E.; Brooks, Matthew J.; Liu, Chunqiao; Bakeri, Hirva A.; Campos, Maria M.; Maubaret, Cecilia; Webster, Andrew R.; Rodriguez, Ignacio R.; Thompson, Debra A.; Bhattacharya, Shomi S.; Koenekoop, Robert K.; Heckenlively, John R.; Swaroop, Anand

    2010-01-01

    Retinal degenerative diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are a leading cause of untreatable blindness with substantive impact on the quality of life of affected individuals and their families. Mouse mutants with retinal dystrophies have provided a valuable resource to discover human disease genes and helped uncover pathways critical for photoreceptor function. Here we show that the rd11 mouse mutant and its allelic strain, B6-JR2845, exhibit rapid photoreceptor dysfunction, followed by degeneration of both rods and cones. Using linkage analysis, we mapped the rd11 locus to mouse chromosome 13. We then identified a one-nucleotide insertion (c.420–421insG) in exon 3 of the Lpcat1 gene. Subsequent screening of this gene in the B6-JR2845 strain revealed a seven-nucleotide deletion (c.14–20delGCCGCGG) in exon 1. Both sequence changes are predicted to result in a frame-shift, leading to premature truncation of the lysophosphatidylcholine acyltransferase-1 (LPCAT1) protein. LPCAT1 (also called AYTL2) is a phospholipid biosynthesis/remodeling enzyme that facilitates the conversion of palmitoyl-lysophosphatidylcholine to dipalmitoylphosphatidylcholine (DPPC). The analysis of retinal lipids from rd11 and B6-JR2845 mice showed substantially reduced DPPC levels compared with C57BL/6J control mice, suggesting a causal link to photoreceptor dysfunction. A follow-up screening of LPCAT1 in retinitis pigmentosa and Leber congenital amaurosis patients did not reveal any obvious disease-causing mutations. Previously, LPCAT1 has been suggested to be critical for the production of lung surfactant phospholipids and biosynthesis of platelet-activating factor in noninflammatory remodeling pathway. Our studies add another dimension to an essential role for LPCAT1 in retinal photoreceptor homeostasis. PMID:20713727

  20. Studies on the Substrate and Stereo/Regioselectivity of Adipose Triglyceride Lipase, Hormone-sensitive Lipase, and Diacylglycerol-O-acyltransferases*

    PubMed Central

    Eichmann, Thomas O.; Kumari, Manju; Haas, Joel T.; Farese, Robert V.; Zimmermann, Robert; Lass, Achim; Zechner, Rudolf

    2012-01-01

    Adipose triglyceride lipase (ATGL) is rate-limiting for the initial step of triacylglycerol (TAG) hydrolysis, generating diacylglycerol (DAG) and fatty acids. DAG exists in three stereochemical isoforms. Here we show that ATGL exhibits a strong preference for the hydrolysis of long-chain fatty acid esters at the sn-2 position of the glycerol backbone. The selectivity of ATGL broadens to the sn-1 position upon stimulation of the enzyme by its co-activator CGI-58. sn-1,3 DAG is the preferred substrate for the consecutive hydrolysis by hormone-sensitive lipase. Interestingly, diacylglycerol-O-acyltransferase 2, present at the endoplasmic reticulum and on lipid droplets, preferentially esterifies sn-1,3 DAG. This suggests that ATGL and diacylglycerol-O-acyltransferase 2 act coordinately in the hydrolysis/re-esterification cycle of TAGs on lipid droplets. Because ATGL preferentially generates sn-1,3 and sn-2,3, it suggests that TAG-derived DAG cannot directly enter phospholipid synthesis or activate protein kinase C without prior isomerization. PMID:23066022

  1. 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

  2. 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.

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

    PubMed

    Wältermann, Marc; Stöveken, Tim; Steinbüchel, Alexander

    2007-02-01

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

  4. 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

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

    PubMed

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

    2011-12-20

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

  6. 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.

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

    PubMed

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

    2016-01-01

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

  8. Direct nonchromatographic assay for 1-acyl-sn-glycerol-3-phosphate acyltransferase

    SciTech Connect

    Rajasekharan, R.; Ray, T.K.; Cronan, J.E. Jr.

    1988-09-01

    1-Acyl-sn-glycerol-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase) which catalyzes the acylation of 1-acyl-sn-glycerol-3-phosphate to phosphatidic acid is generally assayed by the use of a radioactive substrate followed by a time-consuming chromatographic separation of substrate and product. We report a direct and highly sensitive nonchromatographic assay for this enzyme based on the ability of Escherichia coli alkaline phosphatase to dephosphorylate 1-acyl-sn-glycerol-3-phosphate but not phosphatidic acid. This selective hydrolysis coupled with the use of /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate as substrate permits measurement of the product, /sup 32/P-labeled phosphatidic acid by solvent extraction or precipitation. We also report a series of enzymatic reactions for the efficient conversion of /sup 32/Pi to /sup 32/P-labeled 1-acyl-sn-glycerol-3-phosphate.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-11-01

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

  12. 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

  13. Acyl-coenzyme A: cholesterol acyltransferase inhibitor Avasimibe affect survival and proliferation of glioma tumor cell lines.

    PubMed

    Bemlih, Sana; Poirier, Marie-Denise; El Andaloussi, Abdeljabar

    2010-06-15

    Glioblastoma is the most common primary brain tumor in adults and one of its hallmarks is resistance to apoptosis. Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular membrane-bound enzyme that uses cholesterol and long chain fatty acyl-CoA as substrates to produce cholesteryl esters. The presence of cholesteryl esters in glioblastoma may be related to vascular and/or cell neoplastic proliferation in the tumor mass, two prerequisites for tumor cell growth. ACAT activity has been detected in glioblastoma cell homogenates. The present study is the first report on the effect of Avasimibe, a specific inhibitor of ACAT, on glioma cell lines (U87, A172 and GL261). Our results showed that Avasimibe inhibited ACAT-1 expression and cholesterol ester synthesis in glioma cell lines. Moreover, Avasimibe inhibited the growth of the cells by inducing cell cycle arrest and induced apoptosis as a result of caspase-8 and caspase-3 activation. Also, Our findings provide proof of principle that targeting ACAT-1 with the inhibitor Avasimibe could be an efficient therapy in the treatment of glioblastoma.

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

    PubMed

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

    2010-12-03

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

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

    PubMed

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

    2011-01-01

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

  16. 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

  17. 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.

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

    PubMed

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

    2000-01-01

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

  19. Deficiency of glycerol-3-phosphate acyltransferase 1 decreases triacylglycerol storage and induces fatty acid oxidation in insect fat body.

    PubMed

    Alves-Bezerra, Michele; Ramos, Isabela B; De Paula, Iron F; Maya-Monteiro, Clarissa M; Klett, Eric L; Coleman, Rosalind A; Gondim, Katia C

    2017-03-01

    Glycerol-3-phosphate acyltransferases (GPAT) catalyze the initial and rate-limiting step for the de novo synthesis of triacylglycerol (TAG). Four mammalian GPAT isoforms have been identified: the mitochondria-associated GPAT1 and 2, and the endoplasmic reticulum (ER)-associated GPAT3 and 4. In the insect Rhodnius prolixus, a vector of Chagas' disease, we previously predicted a mitochondrial-like isoform (RhoprGPAT1) from genomic data. In the current study, we clone the RhoprGPAT1 coding sequence and identify an ER-associated GPAT (RhoprGPAT4) as the second isoform in the insect. RhoprGPAT1 contributes 15% of the total GPAT activity in anterior midgut, 50% in posterior midgut and fat body, and 70% in the ovary. The RhoprGpat1 gene is the predominant transcript in the midgut and fat body. To evaluate the physiological relevance of RhoprGPAT1, we generate RhoprGPAT1-deficient insects. The knockdown of RhoprGpat1 results in 50% and 65% decrease in TAG content in the posterior midgut and fat body, respectively. RhoprGpat1-deficient insects also exhibits impaired lipid droplet expansion and a 2-fold increase in fatty acid β-oxidation rates in the fat body. We propose that the RhoprGPAT1 mitochondrial-like isoform is required to channel fatty acyl chains towards TAG synthesis and away from β-oxidation. Such a process is crucial for the insect lipid homeostasis.

  20. 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

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

    PubMed

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

    2007-11-13

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

  2. Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25

    SciTech Connect

    Chang, C.C.Y.; Chang, W.; Chang, T.Y. ); Noll, W.W.; Nutile-McMenemy, N. ); Lindsay, E.A.; Baldini, A. )

    1994-01-01

    Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently the authors' laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. They have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human-hamster somatic cell hybrid panels.

  3. Identification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis.

    PubMed

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

    2008-09-01

    Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C(16) and C(18)) or very-long-chain (C(20) and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.

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

    PubMed Central

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

    2008-01-01

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

  5. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    DTIC Science & Technology

    2014-08-01

    to ~20% of melanoma with no effective treatment. Targeting palmitoyl acyltansferases (PATs) involved in N-RAS regulation could be a novel strategy to...treat N-RAS mutant melanoma . The objective of the project is to identify PATs responsible for NRAS activation in melanoma cells using chemical...have carried out PATs profiling in melanoma cells using chemical probes and mRNA profiling. We have identified candidate PATs highly expressed in NRAS

  6. The expression of ghrelin O-acyltransferase (GOAT) in human tissues.

    PubMed

    Lim, Chung Thong; Kola, Blerina; Grossman, Ashley; Korbonits, Márta

    2011-01-01

    Ghrelin is a circulating growth hormone-releasing and appetite-inducing brain-gut peptide. It needs to be acylated on its serine-3 with octanoate for its endocrine actions. The acyl-transferase that catalyses ghrelin octanoylation has recently been identified and named as GOAT (ghrelin O-acyltransferase); GOAT enzyme is coded by the MBOAT4 gene. This study aimed to investigate GOAT expression in the human. The distribution of GOAT mRNA expression was studied in various human tissues using classical and real-time reverse transcription and polymerase chain reaction. GOAT expression was found in all tissues studied (stomach, adrenal cortex, breast, right and left colon, duodenum, jejunum, ileum, fat, Fallopian tube, gallbladder, lymph node, lymphocyte cell line, kidney, liver, lung, muscle, myocardium, pituitary, oesophagus, pancreas, ovary, placenta, prostate, testis, spleen and thyroid). The widespread expression of GOAT corresponds to the widespread distribution of ghrelin expression. GOAT expression was high in stomach and gut, the major ghrelin-secreting tissues, and in the pituitary, in which ghrelin is known to show autocrine and paracrine effects. Identification of GOAT expression in various tissues support the concept that in addition to the important endocrine effect of acylated ghrelin, the paracrine effects of locally synthetised and acylated ghrelin may be important.

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

    PubMed

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

    2013-05-01

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

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

    PubMed

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

    2005-06-01

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

  9. 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

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

    PubMed

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

    2012-01-01

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

  11. Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin*

    PubMed Central

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

    2013-01-01

    The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3cisΔ9,12,15) 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. PMID:23824186

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts.

    PubMed

    Bagnato, Carolina; Igal, R Ariel

    2003-12-26

    Diacylglycerol (DAG) is a versatile molecule that participates as substrate in the synthesis of structural and energetic lipids, and acts as the physiological signal that activates protein kinase C. Diacylglycerol acyltransferase (DGAT), the last committed enzyme in triacylglycerol synthesis, could potentially regulate the content and use of both signaling and glycerolipid substrate DAG by converting it into triacylglycerol. To test this hypothesis, we stably overexpressed the DGAT1 mouse gene in human lung SV40-transformed fibroblasts (DGAT cells), which contains high levels of DAG. DGAT cells exhibited a 3.9-fold higher DGAT activity and a 3.2-fold increase in triacylglycerol content, whereas DAG and phosphatidylcholine decreased by 70 and 20%, respectively, compared with empty vector-transfected SV40 cells (Control cells). Both acylation and de novo synthesis of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were reduced by 30-40% in DGAT cells compared with controls, suggesting that DGAT used substrates for triacylglycerol synthesis that had originally been destined to produce phospholipids. The incorporation of [14C]DAG and [14C]fatty acids released from plasma membrane by additions of either phospholipase C or phospholipase A2 into triacylglycerol was increased by 6.2- and 2.8-fold, respectively, in DGAT cells compared with control cells, indicating that DGAT can attenuate signaling lipids. Finally, DGAT overexpression reversed the neoplastic phenotype because it dramatically reduced the cell growth rate and suppressed the anchorage-independent growth of the SV40 cells. These results strongly support the view that DGAT participates in the regulation of membrane lipid synthesis and lipid signaling, thereby playing an important role in modulating cell growth properties.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2012-02-01

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

  16. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds

    PubMed Central

    Zhang, Chunyu; Iskandarov, Umidjon; Cahoon, Edgar B.

    2013-01-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions. PMID:23814277

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

    PubMed

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

    2015-12-01

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

  18. 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

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

    PubMed Central

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

    2016-01-01

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

  20. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds.

    PubMed

    Zhang, Chunyu; Iskandarov, Umidjon; Klotz, Elliott T; Stevens, Robyn L; Cahoon, Rebecca E; Nazarenus, Tara J; Pereira, Suzette L; Cahoon, Edgar B

    2013-08-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45-50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

  1. 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.

  2. Sequence of the luxD gene encoding acyltransferase of the lux operon from Photobacterium leiognathi.

    PubMed

    Chao, Y F; Weng, S F; Lin, J W

    1993-04-15

    The nucleotide sequence of luxD (EMBL accession No. X65611), encoding acyltransferase (ACT), of the lux operon from Photobacterium leiognathi PL741 was determined, and the amino acid (aa) sequence was deduced. ACT is a component of the fatty acid reductase complex, which is responsible for converting fatty acid to aldehyde that serves as the substrate in the luciferase-catalyzed bioluminescent reactions. The protein has a calculated M(r) of 34,384 and comprises 305 aa residues. Alignment and comparison of the ACT of P. leiognathi with that of Vibrio fischeri ATCC7744, V. harveyi B392 and Xenorhabdus luminescens Hm shows that there is 66%, 59% and 61% aa identity, respectively.

  3. Molecular cloning and expression analysis of porcine ghrelin o-acyltransferase.

    PubMed

    Lin, Tonghui; Meng, Qingyong; Sui, Dandan; Peng, Dezhi; Li, Yang; Liu, Xiaofang; Xie, Longfei; Li, Ning

    2011-10-01

    The peptide hormone ghrelin is secreted in the stomach, with unique N-octanoylation at serine 3, which is a requirement for its functionality. These functions include growth hormone release, appetite stimulation, gastrointestinal motility, glucose regulation, and cell proliferation. The enzyme responsible for ghrelin acylation was recently identified as ghrelin O-acyltransferase (GOAT). In this study, porcine GOAT was cloned and characterized. A full-length cDNA of GOAT of 2013 bp was obtained, which included a 70-bp 5' UTR, a 635-bp 3' UTR, and a 1308-bp open reading frame encoding a protein of 415 amino acids. The GOAT and ghrelin mRNAs are co-expressed in stomach, pancreas, and duodenum at high levels. GOAT was also detected in liver, lung, brain, testis, spleen, kidney, heart, muscle, lipid, and ovary. Our results provide an important basis for further research on GOAT function and the relationship between ghrelin and GOAT.

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

    PubMed Central

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

    2015-01-01

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

  5. Bacterial acyltransferases as an alternative for lipase-catalyzed acylation for the production of oleochemicals and fuels.

    PubMed

    Stöveken, Tim; Steinbüchel, Alexander

    2008-01-01

    Bacterial acyltransferases are a new class of enzymes, and the first member was identified as WS/DGAT in Acinetobacter baylyi ADP1. Their unspecificity have been used in several biotechnological applications for lipid modification, a field that has been dominated by the use of lipases. Examples are the biosynthesis of jojoba-like wax esters and fatty-acid ethyl esters. In addition, these enzymes are also capable of synthesizing acylthioesters. Acyloxoesters and acylthioesters can thus be produced in vivo by whole-cell fermentations rather than in vitro in an enzyme reactor. In this Minireview, we focus on the biotechnological utilization of acyltransferases for the production of modified lipids from renewable resources.

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

    PubMed

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

    2004-03-30

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

  7. RP 64477: a potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase with low systemic bioavailability.

    PubMed

    Bello, A A; Bright, C; Burton, B J; Bush, R C; Casey, J H; Dron, D I; Facchini, V; Joannou, P P; Parrott, D P; Riddell, D; Roberts, S A; Williams, R J

    1996-02-23

    RP 64477 (N-butyl-3-(p-decyloxybenzamido)-4-(methylthio)benzamide) has been shown to be a potent inhibitor of the cholesterol esterifying enzyme Acyl-coenzyme A:cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) in intestinal, hepatic, adrenal, and arterial tissue preparations obtained from a range of animal species. Drug concentrations producing 50% inhibition of enzyme activity (IC50 values) ranged from 14-283 nM. Inhibition by RP 64477 in a rabbit intestinal enzyme preparation was shown to be non-competitive with respect to the substrate oleoyl-CoA. In whole cell assays using human intestinal (CaCo-2), hepatic HepG2) and monocytic (THP-1) cell lines, RP 64477 inhibited ACAT activity with IC50s of 113, 503, and 180 nM, respectively. RP 64477 (0.03% w/w by diet) reduced significantly cholesterol absorption in cholesterol/cholic acid-fed rats from 94+/- 8% to 65 +/- 4%. In cholesterol-fed rabbits, cholesterol absorption was reduced from 72 +/- 5% to 50 +/-5% and 44 +/- 5% at dose levels of 10 and 30 mg kg-1 b.i.d., respectively. Plasma cholesterol levels were reduced dose-dependently in both cholesterol/cholic-acid-fed rats and cholesterol-fed rabbits. Neither cholesterol absorption nor plasma cholesterol levels were reduced significantly in animals maintained on standard laboratory diets. Pharmacokinetic studies indicated that RP 64477 were very poorly absorbed following oral administration to rats. Plasma levels of drug were < 2 ng mL-1 following a dose of 2000 mg kg-1 p.o.. When radiolabelled RP 64477 was administered orally, limited absorption was indicated by the overwhelming elimination of radioactivity in the faces (96.4% of administered material) coupled with low renal clearance (0.6% of dose) and biliary excretion (0.05% of dose). In conclusion, this work shows that RP 64477 is a potent inhibitor of ACAT obtained from a range of animal species and man. Inhibition of cholesterol absorption and hypocholesterolaemic activity has been demonstrated in rats and

  8. A novel erythromycin, 6-desmethyl erythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase.

    PubMed

    Petkovic, Hrvoje; Lill, Rachel E; Sheridan, Rose M; Wilkinson, Barrie; McCormick, Ellen L; McArthur, Hamish A I; Staunton, James; Leadlay, Peter F; Kendrew, Steven G

    2003-06-01

    The acyltransferase (AT) domain in module 4 of the erythromycin polyketide synthase (PKS) was substituted with an AT domain from the rapamycin PKS module 2 in order to alter the substrate specificity from methylmalonyl-CoA to malonyl-CoA. The resulting strain produced 6-desmethyl erythromycin D as the predominant product. This AT domain swap completes the library of malonyl-CoA AT swaps on the erythromycin PKS and reinforces PKS engineering as a robust and generic tool.

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

    PubMed

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

    2010-11-01

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

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

    PubMed

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

    2005-10-15

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

  12. Expression Cloning of a Pseudomonas Gene Encoding a Hydroxydecanoyl-Acyl Carrier Protein-Dependent UDP-GlcNAc Acyltransferase

    PubMed Central

    Dotson, Garry D.; Kaltashov, Igor A.; Cotter, Robert J.; Raetz, Christian R. H.

    1998-01-01

    UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159–5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624–5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is >1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]− 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3′. PMID:9440522

  13. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.

    PubMed

    Chang, C C; Lee, C Y; Chang, E T; Cruz, J C; Levesque, M C; Chang, T Y

    1998-12-25

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.

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

    PubMed

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

    2012-03-14

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

  15. Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii[C][W

    PubMed Central

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

    2012-01-01

    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. PMID:23012436

  16. 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.

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

    PubMed

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

    2015-12-01

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

  18. 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

  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. Ghrelin-ghrelin O-acyltransferase system in the pathogenesis of nonalcoholic fatty liver disease.

    PubMed

    Zhang, Shao-Ren; Fan, Xiao-Ming

    2015-03-21

    Nonalcoholic fatty liver disease (NAFLD) is currently considered as the most common liver disease in Western countries, and is rapidly becoming a serious threat to public health worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not fully understood. The ghrelin-ghrelin O-acyltransferase (GOAT) system has recently been found to play a crucial role in both the development of steatosis and its progression to nonalcoholic steatohepatitis. Ghrelin, the natural ligand of the growth hormone secretagogue receptor, is a 28-amino acid peptide possessing a unique acylation on the serine in position 3 catalyzed by GOAT. The ghrelin-GOAT system is involved in insulin resistance, lipid metabolism dysfunction, and inflammation, all of which play important roles in the pathogenesis of NAFLD. A better understanding of ghrelin-GOAT system biology led to the identification of its potential roles in NAFLD. Molecular targets modulating ghrelin-GOAT levels and the biologic effects are being studied, which provide a new insight into the pathogenesis of NAFLD. This review probes into the possible relationship between the ghrelin-GOAT system and NAFLD, and considers the potential mechanisms by which the ghrelin-GOAT system brings about insulin resistance and other aspects concerning NAFLD.

  1. 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.

  2. Differently Localized Lysophosphatidic Acid Acyltransferases Crucial for Triacylglycerol Biosynthesis in the Oleaginous Alga Nannochloropsis.

    PubMed

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

    2017-02-20

    Production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large amounts of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knockout strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-4), which catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knockouts among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. However, no homologous region has been found with other lipid droplet-associated proteins. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by acquisition of LPATs at the perimeter of lipid droplets. This article is protected by copyright. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2004-09-01

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

  5. Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

    PubMed

    Lamas-Maceiras, Mónica; Vaca, Inmaculada; Rodríguez, Esther; Casqueiro, Javier; Martín, Juan F

    2006-04-01

    A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

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

    PubMed

    Li, L; Pownall, H J

    2000-12-01

    (,Although plasma HDL(2) cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL(2) at a low concentration (< or = 40 microg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL(2) on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL(2) at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL(2), however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL(2) promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL(2) is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.

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

    PubMed

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

    2013-02-01

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

  8. Importance of acyl-coenzyme A:cholesterol acyltransferase 1/2 dual inhibition for anti-atherosclerotic potency of pactimibe.

    PubMed

    Kitayama, Ken; Tanimoto, Tatsuo; Koga, Teiichiro; Terasaka, Naoki; Fujioka, Tomoyuki; Inaba, Toshimori

    2006-07-01

    Pactimibe sulfate, [7-(2,2-dimethylpropanamido)-4,6-dimethyl-1-octylindolin-5-yl]acetic acid hemisulfate, a novel Acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, was investigated in vitro and in vivo to characterize its potential. Pactimibe exhibited dual inhibition for ACAT1 and ACAT2 (concentrations inhibiting 50% [IC50s] at micromolar levels) more potently than avasimibe. Kinetic analysis revealed pactimibe is a noncompetitive inhibitor of oleoyl-CoA (Ki value: 5.6 microM). Furthermore, pactimibe markedly inhibited cholesteryl ester formation (IC50: 6.7 microM) in human monocyte-derived macrophages, and inhibited copper-induced oxidation of low density lipoprotein more potently than probucol. Pactimibe exerted potent lipid-lowering and anti-atherosclerotic effects in atherogenic diet-fed hamsters. At doses of 3 and 10 mg/kg for 90 days, pactimibe decreased serum total cholesterol by 70% and 72%, and aortic fatty streak area by 79% and 95%, respectively. Despite similar cholesterol lowering, fatty streak area reduction was greater by 10 mg/kg. These results suggest that ACAT1/2 dual inhibitor pactimibe has anti-atherosclerotic potential beyond its plasma cholesterol-lowering activity.

  9. Intestine-specific Deletion of Acyl-CoA:Monoacylglycerol Acyltransferase (MGAT) 2 Protects Mice from Diet-induced Obesity and Glucose Intolerance*

    PubMed Central

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

    2014-01-01

    The absorption of dietary fat involves the re-esterification of digested triacylglycerol in the enterocytes, a process catalyzed by acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2. Mice without a functional gene encoding MGAT2 (Mogat2−/−) are protected from diet-induced obesity. Surprisingly, these mice absorb normal amounts of dietary fat but increase their energy expenditure. MGAT2 is expressed in tissues besides intestine, including adipose tissue in both mice and humans. To test the hypothesis that intestinal MGAT2 regulates systemic energy balance, we generated and characterized mice deficient in MGAT2 specifically in the small intestine (Mogat2IKO). We found that, like Mogat2−/− mice, Mogat2IKO mice also showed a delay in fat absorption, a decrease in food intake, and a propensity to use fatty acids as fuel when first exposed to a high fat diet. Mogat2IKO mice increased energy expenditure although to a lesser degree than Mogat2−/− mice and were protected against diet-induced weight gain and associated comorbidities, including hepatic steatosis, hypercholesterolemia, and glucose intolerance. These findings illustrate that intestinal lipid metabolism plays a crucial role in the regulation of systemic energy balance and may be a feasible intervention target. In addition, they suggest that MGAT activity in extraintestinal tissues may also modulate energy metabolism. PMID:24784138

  10. Ablation of Ghrelin O-Acyltransferase Does Not Improve Glucose Intolerance or Body Adiposity in Mice on a Leptin-Deficient ob/ob Background

    PubMed Central

    Kirchner, Henriette; Heppner, Kristy M.; Holland, Jenna; Kabra, Dhiraj; Tschöp, Matthias H.; Pfluger, Paul T.

    2013-01-01

    Type 2 Diabetes is a global health burden and based on current estimates will become an even larger problem in the future. Developing new strategies to prevent and treat diabetes is a scientific challenge of high priority. The stomach hormone ghrelin has been associated with playing a role in the regulation of glucose homeostasis. However, its precise mechanism and impact on whole glucose metabolism remains to be elucidated. This study aims to clarify the role of the two ghrelin isoforms acyl- and desacyl ghrelin in regulating glucose homeostasis. Therefore ghrelin activating enzyme Ghrelin-O-acyltransferase (GOAT) was ablated in leptin-deficient ob/ob mice to study whether specific acyl ghrelin deficiency or desacyl ghrelin abundance modifies glucose tolerance on a massively obese background. As targeted deletion of acyl ghrelin does not improve glucose homeostasis in our GOAT-ob/ob mouse model we conclude that neither acyl ghrelin nor the increased ratio of desacyl/acyl ghrelin is crucial for controlling glucose homeostasis in the here presented model of massive obesity induced by leptin deficiency. PMID:23630616

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

    PubMed

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

    2005-11-01

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

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

    SciTech Connect

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

    2012-08-24

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

  13. The Lunar Configurable Array Telescope (LCAT)

    NASA Astrophysics Data System (ADS)

    Meinel, Aden B.; Meinel, Marjorie P.

    1990-01-01

    The desire for a much larger space telescope than HST by astronomers is clearly demonstrated by the attendance at this Workshop. The reality is that a much larger space telescope than the HST collides with cost scaling reality. Coupled with this reality is the fact that any multi-billion dollar science project must have broad-based support from the science community and solid political support at both Presidential and Congressional levels. The HST successor is certainly in the same multi-billion dollar class as the Super Collider of the physics community, a project that has finally achieved the broad support base necessary for funding to follow. Advocacy of a bigger HST on the general grounds that 'bigger is better' will not be sufficient. A new concept needs to be developed that clearly diverges from scaling up of a traditional HST-type space telescope. With these realities in mind we have a few comments regarding the nature of a possible space telescope that may depart from what the organizers of this Workshop had in mind. The national goal declared by the President is Space Station, the Moon and Mars, in that order. Space Station is a potential location where a large system could be assembled prior to being sent into a high orbit. It is not a desirable environment for a large space telescope. Mars is not relevant as an observatory site. The Moon is very relevant for reasons we will address. Our comments are based on the premise of a permanent Lunar Outpost. One of the main arguments for a lunar telescope is a degree of permanency, that is, as long as a Lunar Outpost is maintained. In contrast, the relatively short lifetime of an orbiting telescope is a disadvantage, especially as a cost penalty. Access to a telescope in a 100,000 km orbit for refurbishment and resupply is a major problem with no solution in the present NASA planning. A telescope in conjunction with a Lunar Outpost means the possibility for continual upgrading or modifying the telescope to meet changing science objectives. The two main technical disadvantages of the Moon are: 1) its gravity field; and 2) direct Sun and Earth light. The gravity term is manageable. It also appears to be feasible to shield the telescope from direct sun and Earth light and from scattering from nearby lunar terrain. Thermal disturbances to the telescope also appear to be manageable by proper shielding, enabling the telescope to become as cold as if it were at a lunar pole crater. If these conditions are met, the telescope could be at a logistically convenient location near the Lunar Outpost. We want to address a concept that is significantly different from those presented in the preliminary communications from Garth Illingworth in order to help fill in the matrix of possibilities. This option, moreover, is of special interest to JPL and could be an area where JPL can contribute in future studies.

  14. The Lunar Configurable Array Telescope (LCAT)

    NASA Technical Reports Server (NTRS)

    Meinel, Aden B.; Meinel, Marjorie P.

    1989-01-01

    The desire for a much larger space telescope than HST by astronomers is clearly demonstrated by the attendance at this Workshop. The reality is that a much larger space telescope than the HST collides with cost scaling reality. Coupled with this reality is the fact that any multi-billion dollar science project must have broad-based support from the science community and solid political support at both Presidential and Congressional levels. The HST successor is certainly in the same multi-billion dollar class as the Super Collider of the physics community, a project that has finally achieved the broad support base necessary for funding to follow. Advocacy of a bigger HST on the general grounds that 'bigger is better' will not be sufficient. A new concept needs to be developed that clearly diverges from scaling up of a traditional HST-type space telescope. With these realities in mind we have a few comments regarding the nature of a possible space telescope that may depart from what the organizers of this Workshop had in mind. The national goal declared by the President is Space Station, the Moon and Mars, in that order. Space Station is a potential location where a large system could be assembled prior to being sent into a high orbit. It is not a desirable environment for a large space telescope. Mars is not relevant as an observatory site. The Moon is very relevant for reasons we will address. Our comments are based on the premise of a permanent Lunar Outpost. One of the main arguments for a lunar telescope is a degree of permanency, that is, as long as a Lunar Outpost is maintained. In contrast, the relatively short lifetime of an orbiting telescope is a disadvantage, especially as a cost penalty. Access to a telescope in a 100,000 km orbit for refurbishment and resupply is a major problem with no solution in the present NASA planning. A telescope in conjunction with a Lunar Outpost means the possibility for continual upgrading or modifying the telescope to meet changing science objectives. The two main technical disadvantages of the Moon are: 1) its gravity field; and 2) direct Sun and Earth light. The gravity term is manageable. It also appears to be feasible to shield the telescope from direct sun and Earth light and from scattering from nearby lunar terrain. Thermal disturbances to the telescope also appear to be manageable by proper shielding, enabling the telescope to become as cold as if it were at a lunar pole crater. If these conditions are met, the telescope could be at a logistically convenient location near the Lunar Outpost. We want to address a concept that is significantly different from those presented in the preliminary communications from Garth Illingworth in order to help fill in the matrix of possibilities. This option, moreover, is of special interest to JPL and could be an area where JPL can contribute in future studies.

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

    PubMed

    Liu, Limin; Gudas, Lorraine J

    2005-12-02

    Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin A deficiency and should be an excellent animal model of vitamin A deficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.

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

    PubMed Central

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

    2017-01-01

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

  17. 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.

  18. Inhibition of ghrelin O-acyltransferase attenuates food deprivation-induced increases in ingestive behavior.

    PubMed

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

    2013-04-01

    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 6h 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 (0-1h), food intake (0-1 and 2-4h), and food hoarding (0-1h and 2 and 3 days) 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.

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

    PubMed

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

    1998-05-01

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

  20. 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

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2013-01-01

    Acyl CoA:diacylglycerol acyltransferase-1 (DGAT-1) catalyzes the final step in triacylglycerol (TAG) synthesis and is highly expressed in the small intestine. Because DGAT-1 knockout mice are resistant to diet-induced obesity, we investigated the acute effects of intragastric (IG) infusion of a small molecule diacylglycerol acyltransferase-1 inhibitor (DGAT-1i) on eating, circulating fat metabolites, indirect calorimetry, and hepatic and intestinal expression of key fat catabolism enzymes in male rats adapted to an 8 h feeding-16 h deprivation schedule. Also, the DGAT-1i effect on fatty acid oxidation (FAO) was investigated in enterocyte cell culture models. IG DGAT-1i infusions reduced energy intake compared with vehicle in high-fat diet (HFD)-fed rats, but scarcely in chow-fed rats. IG DGAT-1i also blunted the postprandial increase in serum TAG and increased β-hydroxybutyrate levels only in HFD-fed rats, in which it lowered the respiratory quotient and increased intestinal, but not hepatic, protein levels of Complex III of the mitochondrial respiratory chain and of mitochondrial hydroxymethylglutaryl-CoA synthase. Finally, the DGAT-1i enhanced FAO in CaCo2 (EC50 = 0.3494) and HuTu80 (EC50 = 0.00762) cells. Thus, pharmacological DGAT-1 inhibition leads to an increase in intestinal FAO and ketogenesis when dietary fat is available. This may contribute to the observed eating-inhibitory effect. PMID:23449193

  4. 12-((5-Iodo-4-azido-2-hydroxybenzoyl)amino)dodecanoic acid: Biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

    SciTech Connect

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

    1990-02-13

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

  5. The transcriptional response of apple alcohol acyltransferase (MdAAT2) to salicylic acid and ethylene is mediated through two apple MYB TFs in transgenic tobacco.

    PubMed

    Li, Peng-Cheng; Yu, Shao-Wei; Shen, Jin; Li, Qing-Qing; Li, Da-Peng; Li, De-Quan; Zheng, Cheng-Chao; Shu, Huai-Rui

    2014-08-01

    Volatile esters are major factors affecting the aroma of apple fruits, and alcohol acyltransferases (AATs) are key enzymes involved in the last steps of ester biosynthesis. The expression of apple AAT (MdAAT2) is known to be induced by salicylic acid (SA) or ethylene in apple fruits, although the mechanism of its transcriptional regulation remains elusive. In this study, we reveal that two apple transcription factors (TFs), MdMYB1 and MdMYB6, are involved in MdAAT2 promoter response to SA and ethylene in transgenic tobacco. According to electrophoretic mobility shift assays, MdMYB1 or MdMYB6 can directly bind in vitro to MYB binding sites in the MdAAT2 promoter. In vivo, overexpression of the two MYB TFs can greatly enhance MdAAT2 promoter activity, as demonstrated by dual luciferase reporter assays in transgenic tobacco. In contrast to the promoter of MdMYB1 or MdMYB6, the MdAAT2 promoter cannot be induced by SA or ethephon (ETH) in transgenic tobacco, even in stigmas in which the MdAAT2 promoter can be highly induced under normal conditions. However, the induced MYB TFs can dramatically enhance MdAAT2 promoter activity under SA or ETH treatment. We conclude that MdMYB1 and MdMYB6 function in MdAAT2 responses to SA and ethylene in transgenic tobacco, suggesting that a similar regulation mechanism may exist in apple.

  6. Hypolipidemic effect of methanol fraction of Aconitum heterophyllum wall ex Royle and the mechanism of action in diet-induced obese rats

    PubMed Central

    Subash, Arun Koorappally; Augustine, Anu

    2012-01-01

    Aconitum heterophyllum is an endangered Himalayan plant included in “lekhaneyagana,” a pharmacological classification mentioned by Charaka in “Charakasamhita” which means reduce excess fat. The subterranean part of the plant is used for the treatment of diseases like nervous system disorders, fever, diarrhea, obesity, etc. In the present study, we are reporting the hypolipidemic effect of methanol fraction of A. heterophyllum. The methanol extract of A. heterophyllum was orally administered in diet-induced obese rats. After four weeks treatment, blood samples were collected for the estimation of serum lipids and lecithin-cholesterol acyltransferase (LCAT). Liver was collected for the assay of HMG-CoA reductase (HMGR). The fecal samples were also collected to estimate the fecal fat content. The A. heterophyllum treatment markedly lowered total cholesterol, triglycerides and apolipoprotein B concentrations in blood serum. It also showed positive effects (increase) on serum high-density lipoprotein cholesterol (HDL-c) and apolipoprotein A1 concentrations. On the other hand, A. heterophyllum treatment lowered HMGR activity, which helps to reduce endogenous cholesterol synthesis and also activated LCAT, helping increase in HDL-c. An increase in fecal fat content is also an indication of the hypolipidemic effect of A. heterophyllum. The significant hypolipidemic effect of A. heterophyllum may be linked to its ability to inhibit HMGR activity and block intestinal fat absorption. The increase in HDL-c may be linked to its ability to activate LCAT enzyme. PMID:23378943

  7. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

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

    PubMed

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

    2017-01-02

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

  9. Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition

    PubMed Central

    Lewin, Tal M.; de Jong, Hendrik; Schwerbrock, Nicole J. M.; Hammond, Linda E.; Watkins, Steven M.; Combs, Terry P.; Coleman, Rosalind A.

    2008-01-01

    Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high sucrose diet or a 3-month high fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1−/− mice contained 20–80% less TAG than the wildtype controls. In addition, hearts from Gpat1−/− mice fed the high-sucrose diet incorporate 60% less [14C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1−/− mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1−/− hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high fat diets and influences the incorporation of 20:4n6 into heart phospholipids. PMID:18522808

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

    PubMed

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

    2016-12-01

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

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

    PubMed

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

    2006-03-01

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

  12. Phylogenetic Analysis of Glycerol 3-Phosphate Acyltransferases in Opisthokonts Reveals Unexpected Ancestral Complexity and Novel Modern Biosynthetic Components

    PubMed Central

    Smart, Heather C.; Mast, Fred D.; Chilije, Maxwell F. J.; Tavassoli, Marjan; Dacks, Joel B.; Zaremberg, Vanina

    2014-01-01

    Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT), have been described, and characterized primarily in model organisms like Saccharomyces cerevisiae and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of ‘fungal’ orthologs in the basal taxa of the holozoa and ‘animal’ orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens. PMID:25340523

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

    PubMed

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

    2009-12-01

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

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

    PubMed

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

    1998-08-01

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

  15. Construction of 4"-isovalerylspiramycin-I-producing strain by in-frame partial deletion of 3-O-acyltransferase gene in Streptomyces spiramyceticus WSJ-1, the bitespiramycin producer.

    PubMed

    Ma, Chunyan; Zhou, Hongxia; Li, Jingyan; Dai, Jianlu; He, Weiqing; Wang, Hongyuan; Wu, Linzhuan; Wang, Yiguang

    2011-01-01

    Bitespiramycin (BT), a multi-component antibiotic consisted mainly of 4"-isovalerylspiramycin I, II and III, is produced by Streptomyces spiramyceticus WSJ-1, a recombinant spiramycin-production strain that harbored the 4"-O-acyltransferase gene (ist) from Streptomyces mycarofaciens 1748, which could isovalerylate the 4"-OH of spiramycin. To eliminate the production of components 4"-isovalerylspiramycin II and III, therefore reducing the component complexity of BT, inactivation of the sspA gene, which encodes the 3-O-acyltransferase responsible for the acylation of spiramycin I to spiramycin II and III, was performed in Streptomyces spiramyceticus WSJ-1, by in-frame partial deletion. The resulting strain, Streptomyces spiramyceticus WSJ-2, is a 4"-isovalerylspiramycin-I-producing strain as expected.

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

    PubMed

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

    2003-05-01

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

  17. Identification of an arylalkylamine N-acyltransferase from Drosophila melanogaster that catalyzes the formation of long-chain N-acylserotonins

    PubMed Central

    Dempsey, Daniel R.; Jeffries, Kristen A.; Anderson, Ryan L.; Carpenter, Anne-Marie; Ospina, Santiago Rodriguez; Merkler, David J.

    2014-01-01

    Arylalkylamine N-acyltransferase-like 22 (AANATL2) from Drosophila melanogaster was expressed and shown to catalyze the formation of long-chain N-acylserotonins and N-acydopamines. Subsequent identification of endogenous amounts of N-acylserotonins and colocalization of these fatty acid amides and AANATL2 transcripts gives supporting evidence that AANATL2 has a role in the biosynthetic formation of these important cell signalling lipids. PMID:24444601

  18. Monoacylglycerols Are Components of Root Waxes and Can Be Produced in the Aerial Cuticle by Ectopic Expression of a Suberin-Associated Acyltransferase1[W][OA

    PubMed Central

    Li, Yonghua; Beisson, Fred; Ohlrogge, John; Pollard, Mike

    2007-01-01

    The interface between plants and the environment is provided for aerial organs by epicuticular waxes that have been extensively studied. By contrast, little is known about the nature, biosynthesis, and role of waxes at the root-rhizosphere interface. Waxes isolated by rapid immersion of Arabidopsis (Arabidopsis thaliana) roots in organic solvents were rich in saturated C18-C22 alkyl esters of p-hydroxycinnamic acids, but also contained significant amounts of both α- and β-isomers of monoacylglycerols with C22 and C24 saturated acyl groups and the corresponding free fatty acids. Production of these compounds in root waxes was positively correlated to the expression of sn-glycerol-3-P acyltransferase5 (GPAT5), a gene encoding an acyltransferase previously shown to be involved in aliphatic suberin synthesis. This suggests a direct metabolic relationship between suberin and some root waxes. Furthermore, when ectopically expressed in Arabidopsis, GPAT5 produced very-long-chain saturated monoacylglycerols and free fatty acids as novel components of cuticular waxes. The crystal morphology of stem waxes was altered and the load of total stem wax compounds was doubled, although the major components typical of the waxes found on wild-type plants decreased. These results strongly suggest that GPAT5 functions in vivo as an acyltransferase to a glycerol-containing acceptor and has access to the same pool of acyl intermediates and/or may be targeted to the same membrane domain as that of wax synthesis in aerial organs. PMID:17496107

  19. 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

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2012-07-13

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

  3. 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.

  4. 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

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

    PubMed

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

    2006-02-01

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

  6. 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.

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

    PubMed

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

    2011-01-01

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

  8. 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.

  9. Mouse lysocardiolipin acyltransferase controls the development of hematopoietic and endothelial lineages during in vitro embryonic stem-cell differentiation

    PubMed Central

    Wang, Chengyan; Faloon, Patrick W.; Tan, Zhijia; Lv, Yaxin; Zhang, Pengbo; Ge, Yu; Deng, Hongkui

    2007-01-01

    The blast colony-forming cell (BL-CFC) was identified as an equivalent to the hemangioblast during in vitro embryonic stem (ES) cell differentiation. However, the molecular mechanisms underlying the generation of the BL-CFC remain largely unknown. Here we report the isolation of mouse lysocardiolipin acyltransferase (Lycat) based on homology to zebrafish lycat, a candidate gene for the cloche locus. Mouse Lycat is expressed in hematopoietic organs and is enriched in the Lin−C-Kit+Sca-1+ hematopoietic stem cells in bone marrow and in the Flk1+/hCD4+(Scl+) hemangioblast population in embryoid bodies. The forced Lycat transgene leads to increased messenger RNA expression of hematopoietic and endothelial genes as well as increased blast colonies and their progenies, endothelial and hematopoietic lineages. The Lycat small interfering RNA transgene leads to a decrease expression of hematopoietic and endothelial genes. An unbiased genomewide microarray analysis further substantiates that the forced Lycat transgene specifically up-regulates a set of genes related to hemangioblasts and hematopoietic and endothelial lineages. Therefore, mouse Lycat plays an important role in the early specification of hematopoietic and endothelial cells, probably acting at the level of the hemangioblast. PMID:17675553

  10. 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.

  11. 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.

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

    PubMed Central

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

    2016-01-01

    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

  13. Glycerol-3-Phosphate Acyltransferase Contributes to Triacylglycerol Biosynthesis, Lipid Droplet Formation, and Host Invasion in Metarhizium robertsii

    PubMed Central

    Gao, Qiang; Shang, Yanfang; Huang, Wei

    2013-01-01

    Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals. Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting step in glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhizium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene deletion mutant is viable. Compared to the wild type and the gene-rescued mutant, the ΔmrGAT mutant demonstrated reduced abilities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized to the endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results showed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study not only advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii can serve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine the mechanistic connection of GPAT to LD formation. PMID:24077712

  14. In silico characterization of 1,2-diacylglycerol cholinephosphotransferase and lysophospha-tidylcholine acyltransferase genes in Glycine max L. Merrill.

    PubMed

    Sousa, C S; Barros, B A; Barh, D; Ghosh, P; Azevedo, V; Barros, E G; Moreira, M A

    2016-08-26

    The enzymes 1,2-diacylglycerol cholinephosphotrans-ferase (CPT) and lysophosphatidylcholine acyltransferase (LPCAT) are important in lipid metabolism in soybean seeds. Thus, understand-ing the genes that encode these enzymes may enable their modification and aid the improvement of soybean oil quality. In soybean, the genes encoding these enzymes have not been completely described; there-fore, this study aimed to identify, characterize, and analyze the in silico expression of these genes in soybean. We identified two gene models encoding CPT and two gene models encoding LPCAT, one of which presented an alternative transcript. The sequences were positioned on the physical map of soybean and the promoter regions were analyzed. Cis-elements responsible for seed-specific expression and responses to biotic and abiotic stresses were identified. Virtual expression analysis of the gene models for CPT and LPCAT indicated that these genes are expressed under different stress conditions, in somatic embryos during differentiation, in immature seeds, root tissues, and calli. Putative ami-no acid sequences revealed the presence of transmembrane domains, and analysis of the cellular localization of these enzymes revealed they are located in the endoplasmic reticulum.

  15. Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

    PubMed Central

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

    1990-01-01

    A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied. PMID:2310381

  16. 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

  17. 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

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

    DTIC Science & Technology

    2007-04-01

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

  19. [Deletion of spiramycin 3-O-acyltransferase gene from Streptomyces spiramyceticus F21 resulting in the production of spiramycin I as major component].

    PubMed

    Wu, Lin-Zhuan; Ma, Chun-Yan; Wang, Yi-Guang; Dai, Jian-Lu; Li, Jing-Yan; Xia, Huan-Zhang

    2007-07-01

    Spiramycin (SP) belongs to the 16-member macrolide antibiotics. It contains three components,namely SP I, SP II and SP III, which differ structurally in the acylation moieties on the C3 of the lactone. The SP I component contains a hydroxyl group at C3. SP II, and SP III are formed by further acetylation or propionylation of the C3 of SP I, by the same 3-O-acyltransferase (3-O-AT) . The study focused on simplifying spiramycin components. Theoretically, disruption/deletion of the 3-O-AT gene will reduce/stop the acylation of SP I to SP II and SP III. In this study, degenerated primers were designed according to the conserved regions of 3-O-acyltransferase, MdmB and AcyA in the medicamycin and carbomycin producers of S. mycarofaciens and S. thermotolerans, respectively, and an 878bp DNA fragment was amplified from the spiramycin-producer of S. spiramyceticus F21. Blast analysis of the 878bp DNA fragment suggested that it encoded the 3-O-acyltransferase (3-0-AT, sspA) gene for spiramycin biosynthesis. The flanking regions of this 878bp DNA fragment were then amplified by single-oligonucleotide-nested PCR, and a total of 4.3 kb DNA was obtained (3457nt among the 4.3kb fragment was sequenced, and deposited in GenBank DQ642742),covering the whole putative 3-O-acyltransferase gene, sspA. The sspA was then deleted from the S. spiramyceticus F21 genome by double cross-over homologous recombination, mediated by temperature-sensitive plasmid pKC1139. A comparison was done of the components of spiramycins produced by the sspA-deleted mutant strain with that of the parent strain by HPLC analysis, which showed that sspA-deleted mutant produced SP I (72%), SP II (18%), and SP III (9.6%), whereas parent strain produced SP I (7.8%), SP II (67%), and SP III (25%), respectively, demonstrating the role of ssp A in the acylation of SP I into SP II and SP III. The ssp A-deleted mutant strain obtained in this study may be used for the production of SP I, or may serve as a good starter

  20. 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

  1. Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation1[W][OPEN

    PubMed Central

    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

  2. Comparative Analysis of the Substrate Specificity of trans- versus cis-Acyltransferases of Assembly Line Polyketide Synthases

    PubMed Central

    2015-01-01

    Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides. PMID:24871074

  3. 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.

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

    PubMed

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

    2015-11-01

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

  5. 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

  6. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    PubMed Central

    Bansal, Sunil; Durrett, Timothy P.

    2016-01-01

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

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

    PubMed

    Dennis, Jennifer F; Kurosaka, Hiroshi; Iulianella, Angelo; Pace, Jennifer; Thomas, Nancy; Beckham, Sharon; Williams, Trevor; Trainor, Paul A

    2012-01-01

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

  8. Dual Role for Phospholipid:Diacylglycerol Acyltransferase: Enhancing Fatty Acid Synthesis and Diverting Fatty Acids from Membrane Lipids to Triacylglycerol in Arabidopsis Leaves[C][W

    PubMed Central

    Fan, Jilian; Yan, Chengshi; Zhang, Xuebin; Xu, Changcheng

    2013-01-01

    There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass. PMID:24076979

  9. Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii.

    PubMed Central

    Pagot, Y; Belin, J M

    1996-01-01

    This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii. The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate. The subcellular sites and induction patterns were studied. The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo. We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates. PMID:8837442

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

    PubMed

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

    2015-08-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

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

    PubMed

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

    2001-04-30

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

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

    PubMed Central

    2011-01-01

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

  14. PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

    PubMed

    Lopez, Adam M; Chuang, Jen-Chieh; Posey, Kenneth S; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L; Turley, Stephen D

    2015-11-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management.

  15. PRD125, a Potent and Selective Inhibitor of Sterol O-Acyltransferase 2 Markedly Reduces Hepatic Cholesteryl Ester Accumulation and Improves Liver Function in Lysosomal Acid Lipase-Deficient Mice

    PubMed Central

    Lopez, Adam M.; Chuang, Jen-Chieh; Posey, Kenneth S.; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L.

    2015-01-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal−/− mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal+/+ littermates (23 versus 1.8 mg, respectively). In Lal−/− males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal−/− mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management. PMID:26283692

  16. Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver.

    PubMed

    Lopez, Adam M; Posey, Kenneth S; Turley, Stephen D

    2014-11-07

    Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal(-)(/)(-):Soat2(+)(/)(+) mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9mg in Lal(+/+):Soat2(+/+) littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal(-)(/)(-):Soat2(+)(/)(+) mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal(-)(/)(-):Soat2(-)(/)(-) littermates. The level of EC accumulation in the SI of the Lal(-)(/)(-):Soat2(-)(/)(-) mice was also much less than in their Lal(-)(/)(-):Soat2(+)(/)(+) littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal(-)(/)(-):Soat2(-)(/)(-) mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.

  17. Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry.

    PubMed Central

    Aplin, R T; Baldwin, J E; Roach, P L; Robinson, C V; Schofield, C J

    1993-01-01

    Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. Images Figure 1 PMID:8396910

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

    PubMed Central

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

    2016-01-01

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

  19. Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases*

    PubMed Central

    Lemonidis, Kimon; Sanchez-Perez, Maria C.; Chamberlain, Luke H.

    2015-01-01

    S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7–24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs. PMID:26198635

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

    PubMed

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

    2016-01-01

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

  1. Stimualtion of glycerolphosphate phosphatidyltransferase activity in fetal rabbit lung by cortisol administration.

    PubMed

    Rooney, S A; Gross, I; Gassenheimer, L N; Motoyama, E K

    1975-09-19

    Phosphatidylglycerol is an important component of pulmonary surfactant. Previous studies have shown that direct administration of corticosteroids of thyroxine to the fetus during the latter part of gestation results in accelerated lung maturation with increased surfactant production. We have shown that administration of cortisol to fetal rabbits at 24 days' gestation results 3 days later in a significant increase in the activity of pulmonary glycerolphosphate phosphatidyltransferase, an enzyme involved in the synthesis of phosphatidylglycerol. The activity of the liver enzyme was not affected. Choline phosphotransferase, CDPdiglyceride-inositol phosphatidyltransferase, lysophosphatidic acid acyltransferase and lysolecithin acyltransferase activities were not altered significantly by cortisol treatment. Thyroxine treatment had no effect on any of the enzymes of phospholipid or fatty acid biosynthesis studied.

  2. Deficient Cholesterol Esterification in Plasma of apoc2 Knockout Zebrafish and Familial Chylomicronemia Patients

    PubMed Central

    Liu, Chao; Gaudet, Daniel; Miller, Yury I.

    2017-01-01

    Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Apolipoprotein C-II (APOC2) is an obligatory cofactor for lipoprotein lipase (LPL), the major enzyme catalyzing plasma triglyceride hydrolysis. We have created an apoc2 knockout zebrafish model, which mimics the familial chylomicronemia syndrome (FCS) in human patients with a defect in the APOC2 or LPL gene. In this study, we measured plasma levels of free cholesterol (FC) and cholesterol esters (CE) and found that apoc2 mutant zebrafish have a significantly higher FC to CE ratio (FC/CE), when compared to the wild type. Feeding apoc2 mutant zebrafish a low-fat diet reduced triglyceride levels but not the FC/CE ratio. In situ hybridization and qPCR results demonstrated that the hepatic expression of lecithin-cholesterol acyltransferase (lcat), the enzyme responsible for esterifying plasma FC to CE, and of apolipoprotein A-I, a major protein component of HDL, were dramatically decreased in apoc2 mutants. Furthermore, the FC/CE ratio was significantly increased in the whole plasma and in a chylomicron-depleted fraction of human FCS patients. The FCS plasma LCAT activity was significantly lower than that of healthy controls. In summary, this study, using a zebrafish model and human patient samples, reports for the first time the defect in plasma cholesterol esterification associated with LPL deficiency. PMID:28107429

  3. Dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), alters plasma high-density lipoprotein-cholesterol levels and hepatic gene expression in rats.

    PubMed

    Aizawa, Koichi; Inakuma, Takahiro

    2009-12-01

    The effects of dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), on lipid metabolism were examined. Young male Wistar rats were fed diets containing paprika powder, paprika organic solvent extract, residue of paprika extract, and purified capsanthin. Administration of purified capsanthin for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P < 0.05) without detectable differences in plasma total cholesterol and TAG concentrations. A statistically significant correlation (r 0.567; P < 0.001) was found between dietary capsanthin concentrations and plasma HDL-cholesterol concentrations. Animals receiving diets containing two different capsanthin concentrations exhibited dose-dependent increases in plasma HDL-cholesterol (r 0.597; P < 0.005). While capsanthin was absent in the liver of animals fed the basal diet, it increased markedly in capsanthin-fed animals (P < 0.001). Quantitative analyses of hepatic mRNA levels revealed that capsanthin administration resulted in up-regulation of mRNA for apoA5 and lecithin cholesterol acyltransferase (LCAT), without significant differences in other mRNA levels related to HDL-cholesterol metabolism. These results suggest that capsanthin had an HDL-cholesterol-raising effect on plasma, and the potential to increase cholesterol efflux to HDL particles by increasing apoA5 levels and/or enhancement of LCAT activity.

  4. Fetal macrosomia related to maternal poorly controlled type 1 diabetes strongly impairs serum lipoprotein concentrations and composition

    PubMed Central

    Merzouk, H; Bouchenak, M; Loukidi, B; Madani, S; Prost, J; Belleville, J

    2000-01-01

    Aims—To determine the effects of fetal macrosomia related to maternal type 1 diabetes on the lipid transport system. Methods—Serum lipoprotein concentrations and composition and lecithin:cholesterol acyltransferase (LCAT) activity were investigated in macrosomic newborns (mean birth weight, 4650 g; SEM, 90) and their mothers with poorly controlled type 1 diabetes, in appropriate for gestational age newborns (mean birth weight, 3616 g; SEM, 68) and their mothers with well controlled type 1 diabetes, and macrosomic (mean birth weight, 4555 g; SEM, 86) or appropriate for gestational age (mean birth weight, 3290 g; SEM, 45) newborns and their healthy mothers. Results—In mothers with well controlled type 1 diabetes, serum lipids, apolipoproteins, and lipoproteins were comparable with those of healthy mothers. Similarly, in their infants, these parameters did not differ from those of appropriate for gestational age newborns. Serum triglyceride, very low density lipoprotein (VLDL), apolipoprotein B100 (apo B100), and high density lipoprotein (HDL) triglyceride concentrations were higher, whereas serum apo A-I and HDL3 concentrations were lower in mothers with diabetes and poor glycaemic control than in healthy mothers. Their macrosomic newborns had higher concentrations in all serum lipids and lipoproteins, with high apo A-I and apo B100 values compared with appropriate for gestational age newborns. In macrosomic infants of healthy mothers, there were no significant differences in lipoprotein profiles compared with those of appropriate for gestational age infants. LCAT activity was similar in both groups of mothers and newborns. Conclusion—Poorly controlled maternal type 1 diabetes and fetal macrosomia were associated with lipoprotein abnormalities. Macrosomic lipoprotein profiles related to poor metabolic control of type 1 diabetes appear to have implications for later metabolic diseases. Key Words: apolipoproteins • lipids • lipoproteins • lecithin

  5. A strategy for quantitative bioanalysis of non-polar neutral compounds by liquid chromatography/electrospray ionization tandem mass spectrometry: determination of TS-962, a novel acyl-CoA:cholesterol acyltransferase inhibitor, in rabbit aorta and liver tissues.

    PubMed

    Yamaguchi, J; Matsuno, Y; Hachiuma, K; Ogawa, N; Higuchi, S

    2001-01-01

    A strategy for the sensitive and reliable quantitative determination of non-polar neutral compounds in biological matrices by liquid chromatography/electrospray ionization tandem mass spectrometry is described in the context of assay development for TS-962, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, in rabbit aorta and liver tissues. The electrospray ionization (ESI) mass spectrum of this compound with a mobile phase of water/acetonitrile did not give abundant [M + H]+ ions, but did give alkali metal cation adducts such as [M + Na]+, [M + CH3CN + Na]+ and [M + K]+ ions. The cationized species are acknowledged as unsuitable precursor ions for selected reaction monitoring (SRM) for various reasons, such as difficulty in obtaining characteristic product ions in low-energy collision-induced dissociation, and irreproducibility of the adduct-ion intensities. To overcome this problem, a solution of 3.4 mM trifluoroacetic acid in 2-propanol was added to the mobile phase as a postcolumn additive, resulting in a decrease of the undesirable adduct formation and significant enhancement of [M + H]+ ion intensity. An attempt was then made to prevent the matrix effect by employing a column-switching system, which allowed direct injection of a large volume of 2-propanolic tissue homogenate (950 microL) followed by sufficient clean-up, separation, and ESI-SRM on-line. This enabled development of a sensitive and reliable assay method for TS-962 in rabbit aorta and liver tissues in the concentration range of 5-500 ng/g wet tissue using a 25-mg aliquot of tissue sample. Application of this method to the determination of aortic TS-962 levels at 24 h after repeated oral administration of this compound (3 mg/kg) once a day for 12 weeks to 1% cholesterol-fed rabbits is also presented. Results showed that TS-962 is well distributed to both the thoracic and abdominal aorta tissues, at levels higher than the 50% inhibitory concentration value of this compound for

  6. Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels

    PubMed

    Knutzon; Hayes; Wyrick; Xiong; Maelor Davies H; Voelker

    1999-07-01

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

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

    PubMed Central

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

    2014-01-01

    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. PMID:24909748

  8. 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

  9. 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.

  10. Inhibition of acyl-CoA cholesterol O-acyltransferase reduces the cholesteryl ester enrichment of atherosclerotic lesions in the Yucatan micropig.

    PubMed

    Bocan, T M; Mueller, S B; Uhlendorf, P D; Brown, E Q; Mazur, M J; Black, A E

    1993-03-01

    Atherosclerotic lesion development may be altered indirectly by regulating plasma cholesterol or directly by inhibition of acyl-CoA cholesterol O-acyltransferase (ACAT) within cells of the artery. Yucatan micropigs were meal-fed a 2% cholesterol, 8% peanut oil, 8% coconut oil purified diet for 1 month prior to administration of the potent, bioavailable ACAT inhibitor CI-976, and induction of atherosclerotic lesions by chronic endothelial damage. After 84-108 days of therapy, CI-976 decreased mean plasma VLDL-cholesterol 85-91% and cumulative VLDL-exposure (area under VLDL-time curve) by 65%. However, overall plasma total, LDL and HDL cholesterol and triglyceride levels were unchanged. CI-976 decreased liver cholesteryl ester (CE) content 65% without significantly affecting adrenal CE content. The CE content of the injured left femoral, left iliac and abdominal aorta and uninjured right femoral and iliac arteries and thoracic aorta was reduced 62-78% by CI-976. Systemic plasma CI-976 levels measured 24 h post-dose ranged from 2.26 to 4.05 micrograms/ml and significantly correlated with the reduction in both VLDL and vessel CE content. Thus, we conclude that inhibition of ACAT can blunt the cholesteryl ester enrichment of developing atherosclerotic lesions by preventing reesterification and storage of lipoprotein cholesterol within vascular cells and by reducing the plasma level and delivery to the arterial wall of such atherogenic lipoproteins as VLDL.

  11. Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase

    PubMed Central

    Callahan, Damien L.; Dubois, David; van Dooren, Giel G.; Shears, Melanie J.; Cesbron-Delauw, Marie-France; Maréchal, Eric; McConville, Malcolm J.; McFadden, Geoffrey I.; Yamaryo-Botté, Yoshiki; Botté, Cyrille Y.

    2016-01-01

    Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii. PMID:27490259

  12. 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

  13. The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing.

    PubMed

    García-Estrada, Carlos; Vaca, Inmaculada; Fierro, Francisco; Sjollema, Klaas; Veenhuis, Marten; Martín, Juan Francisco

    2008-06-01

    Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (beta subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.

  14. The effects of sterol structure upon sterol esterification.

    PubMed

    Lin, Don S; Steiner, Robert D; Merkens, Louise S; Pappu, Anuradha S; Connor, William E

    2010-01-01

    Cholesterol is esterified in mammals by two enzymes: LCAT (lecithin cholesterol acyltransferase) in plasma and ACAT(1) and ACAT(2) (acyl-CoA cholesterol acyltransferases) in the tissues. We hypothesized that the sterol structure may have significant effects on the outcome of esterification by these enzymes. To test this hypothesis, we analyzed sterol esters in plasma and tissues in patients having non-cholesterol sterols (sitosterolemia and Smith-Lemli-Opitz syndrome). The esterification of a given sterol was defined as the sterol ester percentage of total sterols. The esterification of cholesterol in plasma by LCAT was 67% and in tissues by ACAT was 64%. Esterification of nine sterols (cholesterol, cholestanol, campesterol, stigmasterol, sitosterol, campestanol, sitostanol, 7-dehydrocholesterol and 8-dehydrocholesterol) was examined. The relative esterification (cholesterol being 1.0) of these sterols by the plasma LCAT was 1.00, 0.95, 0.89, 0.40, 0.85, 0.82 and 0.80, 0.69 and 0.82, respectively. The esterification by the tissue ACAT was 1.00, 1.29, 0.75, 0.49, 0.45, 1.21 and 0.74, respectively. The predominant fatty acid of the sterol esters was linoleic acid for LCAT and oleic acid for ACAT. We compared the esterification of two sterols differing by only one functional group (a chemical group attached to sterol nucleus) and were able to quantify the effects of individual functional groups on sterol esterification. The saturation of the A ring of cholesterol increased ester formation by ACAT by 29% and decreased the esterification by LCAT by 5.9%. Esterification by ACAT and LCAT was reduced, respectively, by 25 and 11% by the presence of an additional methyl group on the side chain of cholesterol at the C-24 position. This data supports our hypothesis that the structure of the sterol substrate has a significant effect on its esterification by ACAT or LCAT.

  15. Biological Activities of 2-Mercaptobenzothiazole Derivatives: A Review

    PubMed Central

    Azam, Mohammed Afzal; Suresh, Bhojraj

    2012-01-01

    2-Mercaptobenzothiazoles are an important class of bioactive and industrially important organic compounds. These compounds are reported for their antimicrobial and antifungal activities, and are subsequently highlighted as a potent mechanism-based inhibitor of several enzymes like acyl coenzyme A cholesterol acyltransferase, monoamine oxidase, heat shock protein 90, cathepsin D, and c-Jun N-terminal kinases. These derivatives are also known to possess antitubercular, anti-inflammatory, antitumor, amoebic, antiparkinsonian, anthelmintic, antihypertensive, antihyperlipidemic, antiulcer, chemoprotective, and selective CCR3 receptor antagonist activity. This present review article focuses on the pharmacological profile of 2-mercaptobenzothiazoles with their potential activities. PMID:23264933

  16. 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

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

    PubMed

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

    2004-11-01

    This study tested the hypothesis that avasimibe, an inhibitor of acyl coenzyme A-cholesterol acyltransferase (ACAT), would improve treadmill exercise performance in patients with claudication secondary to peripheral arterial disease (PAD). Four hundred and forty-two patients with PAD (ankle-brachial index in the index leg of < or =0.90 with a > or =20% reduction post-exercise) were enrolled from 39 centers in the USA. Patients were randomized to receive oral avasimibe 50 mg, 250 mg, 750 mg or placebo for a treatment period of 12 months. Changes from baseline in peak walking time (PWT) using a graded treadmill protocol were compared among groups after 6 and 12 months of treatment. Individual group comparisons were considered statistically significant if p < 0.0245 for the 50 mg and 250 mg groups and p < 0.001 for the 750 mg group. Patients randomized to the 50 mg group experienced a 0.76 min net increase over placebo in PWT, but this did not reach the pre-specified level of statistical significance (Hochberg procedure p = 0.027) using ANCOVA after 12 months of treatment after adjusting for multiple comparisons. This trend in PWT was supported by the changes in treadmill initial claudication time (ICT) (p = 0.026) and Walking Impairment Questionnaire (WIQ) walking distance score (p = 0.058). The 250 mg and 750 mg avasimibe dose groups failed to demonstrate an improvement in PWT over placebo after 6 months of treatment. In conclusion, while the ACAT inhibitor avasimibe did not show clear evidence of benefit on treadmill exercise performance in patients with PAD, the results add to our knowledge of the impact of treatments directed at atherosclerosis on functional endpoints.

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

    PubMed

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

    2009-06-01

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

  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.

  20. Adult-onset deficiency of acyl CoA:monoacylglycerol acyltransferase 2 protects mice from diet-induced obesity and glucose intolerance[S

    PubMed Central

    Banh, Taylor; Nelson, David W.; Gao, Yu; Huang, Ting-Ni; Yen, Mei-I; Yen, Chi-Liang E.

    2015-01-01

    Acyl-CoA:monoacylglycerol acyltransferase (MGAT) 2 catalyzes triacylglycerol (TAG) synthesis, required in intestinal fat absorption. We previously demonstrated that mice without a functional MGAT2-coding gene (Mogat2−/−) exhibit increased energy expenditure and resistance to obesity induced by excess calories. One critical question raised is whether lacking MGAT2 during early development is required for the metabolic phenotypes in adult mice. In this study, we found that Mogat2−/− pups grew slower than wild-type littermates during the suckling period. To determine whether inactivating MGAT2 in adult mice is sufficient to confer resistance to diet-induced obesity, we generated mice with an inducible Mogat2-inactivating mutation. Mice with adult-onset MGAT2 deficiency (Mogat2AKO) exhibited a transient decrease in food intake like Mogat2−/− mice when fed a high-fat diet and a moderate increase in energy expenditure after acclimatization. They gained less weight than littermate controls, but the difference was smaller than that between wild-type and Mogat2−/− mice. The moderate reduction in weight gain was associated with reduced hepatic TAG and improved glucose tolerance. Similar protective effects were also observed in mice that had gained weight on a high-fat diet before inactivating MGAT2. These findings suggest that adult-onset MGAT2 deficiency mitigates metabolic disorders induced by high-fat feeding and that MGAT2 modulates early postnatal nutrition and may program metabolism later in life. PMID:25535286

  1. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  2. Fatty acyltranferases in serum in cystic fibrosis (CF) patients

    SciTech Connect

    Zielenski, J.; Newman, L.J.; Slomiany, B.L.; Slomiany, A.

    1987-05-01

    Studies on serum and gastrointestinal secretion from CF patient is suggest that defective accumulation of mucus in gastrointestinal tract and excessive amount of a protease resistant peptides in serum are related to the abnormal activity of enzymes responsible for fatty acylation of proteins. Here, the authors investigated the fatty acyltransferase activities in serum of normal and CF patients. A 15 l of serum was mixed with 0.85 nmol ( UC)palmitoyl CoA, 200 g of serine and threonine and incubated at 37C for 30 min. The incubates were immediately frozen, dried extracted with C/M and chromatographed in chloroform/methanol/water. The incorporation of ( UC)palmitate was determined using linear radioscanner and authoradiography. The results of HPTLC revealed that CF serum in addition of ACAT and LCAT contained enzymes responsible for the transfer of ( UC)palmitate to monoacylphosphoglycerides, and serine and threonine. In normal serum the formation of a small amount of palmitoyl serine and palmitoyl threonine was also observed but the acylation of monoacylphosphoglycerides was not detectable. The authors conclude that in cystic fibrosis the abnormal fatty acyltransferases are responsible for the occurrence of protease resistant glycoprotein, unusual peptides in serum and possibly for the modification of membrane proteins and lipids.

  3. 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

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

    PubMed Central

    2010-01-01

    Background The association of rs1044925 polymorphism in the acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene and serum lipid profiles is not well known in different ethnic groups. Bai Ku Yao is a special subgroup of the Yao minority in China. The present study was carried out to clarify the association of rs1044925 polymorphism in the ACAT-1 gene and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 626 subjects of Bai Ku Yao and 624 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples. Genotyping of rs1044925 polymorphism in the ACAT-1 gene was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Results The levels of serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), apolipoprotein (Apo) AI and ApoB were lower in Bai Ku Yao than in Han (P < 0.01 for all). The frequency of A and C alleles was 79.0% and 21.0% in Bai Ku Yao, and 87.3% and 12.7% in Han (P < 0.001); respectively. The frequency of AA, AC and CC genotypes was 63.2%, 31.4% and 5.2% in Bai Ku Yao, and 75.6%, 23.2% and 1.1% in Han (P < 0.001); respectively. The levels of TC, LDL-C and ApoB in Bai Ku Yao but not in Han were different between the AA and AC/CC genotypes in females but not in males (P < 0.05 for all). The C allele carriers had lower serum TC, LDL-C and ApoB levels as compared with the C allele noncarriers. The levels of TC, LDL-C and ApoB in Bai Ku Yao but not in Han were correlated with genotypes in females but not in males (P < 0.05 for all). Serum lipid parameters were also correlated with sex, age, body mass index, alcohol consumption, and blood pressure in both ethnic groups (P < 0.05-0.001). Conclusions These results suggest that the polymorphism of rs1044925 in the ACAT-1 gene is mainly associated with female serum TC, LDL-C and

  5. Effect of diacylglycerol acyltransferase 2 overexpression in 3T3-L1 is associated to an increase in mono-unsaturated fatty acid accumulation

    PubMed Central

    2014-01-01

    Background Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells. Methods To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a 13C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography–mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d. Results The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotope-labeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA

  6. 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

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

    PubMed

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

    2013-04-01

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

  8. Response of the cholesterol metabolism to a negative energy balance in dairy cows depends on the lactational stage.

    PubMed

    Gross, Josef J; Kessler, Evelyne C; Albrecht, Christiane; Bruckmaier, Rupert M

    2015-01-01

    The response of cholesterol metabolism to a negative energy balance (NEB) induced by feed restriction for 3 weeks starting at 100 days in milk (DIM) compared to the physiologically occurring NEB in week 1 postpartum (p.p.) was investigated in 50 dairy cows (25 control (CON) and 25 feed-restricted (RES)). Blood samples, liver biopsies and milk samples were taken in week 1 p.p., and in weeks 0 and 3 of feed restriction. Plasma concentrations of total cholesterol (C), phospholipids (PL), triglycerides (TAG), very low density lipoprotein-cholesterol (VLDL-C) and low density lipoprotein-cholesterol (LDL-C) increased in RES cows from week 0 to 3 during feed restriction and were higher in week 3 compared to CON cows. In contrast, during the physiologically occurring NEB in week 1 p.p., C, PL, TAG and lipoprotein concentrations were at a minimum. Plasma phospholipid transfer protein (PLTP) and lecithin:cholesterol acyltransferase (LCAT) activities did not differ between week 0 and 3 for both groups, whereas during NEB in week 1 p.p. PLTP activity was increased and LCAT activity was decreased. Milk C concentration was not affected by feed restriction in both groups, whereas milk C mass was decreased in week 3 for RES cows. In comparison, C concentration and mass in milk were elevated in week 1 p.p. Hepatic mRNA abundance of sterol regulatory element-binding factor-2 (SREBF-2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), and ATP-binding cassette transporter (ABCA1) were similar in CON and RES cows during feed restriction, but were upregulated during NEB in week 1 p.p. compared to the non-lactating stage without a NEB. In conclusion, cholesterol metabolism in dairy cows is affected by nutrient and energy deficiency depending on the stage of lactation.

  9. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

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

    PubMed Central

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

    2010-01-01

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications. PMID:20439724

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

    PubMed

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

    2006-02-01

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

  12. Reassessing the Potential Activities of Plant CGI-58 Protein.

    PubMed

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

  13. Reassessing the Potential Activities of Plant CGI-58 Protein

    PubMed Central

    Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

    2016-01-01

    Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

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

    PubMed

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

    2015-03-01

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

  15. Activities.

    ERIC Educational Resources Information Center

    Bippert, Judy

    1993-01-01

    Presents activities designed to give students an opportunity to solve concrete problems involving spatial relationships and logical thinking utilizing hands-on manipulatives. Provides teacher instructions and four reproducible worksheets. (MDH)

  16. The mouse plasma PAF acetylhydrolase: I. Characterization and properties.

    PubMed

    Tsaoussis, V; Vakirtzi-Lemonias, C

    1994-05-01

    Mouse plasma platelet-activating factor acetylhydrolase (PAF-AH) has an apparent Km of 7.4 microM and a Vmax of 21.6 nmol/min per mg protein. Comparison with values reported for the human and the rat enzymes shows at least a 5-fold higher Vmax and similar enzyme-substrate affinity. Although lecithin:cholesterol acyltransferase (LCAT) and one component of the PAF-AH share similar masses and lipoprotein association, they are distinct enzymes. Similarly, PAF-AH is distinct from the phospholipase A2 (PLA2) and the lysophospholipase of mouse plasma. A series of PAF structural analogs showed either competitive inhibition or a mixed type of inhibition of PAF-AH. Mouse plasma PAF-AH is highly sensitive to 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and is activated by deoxycholate. SDS-PAGE showed that two distinct proteins with molecular masses of 46 and 63 kDa contribute to the PAF-AH activity. The HDL-VHDL lipoprotein associated PAF-AH is precipitated to an extent of about 60% by phosphotungstate-MgCl2 and Tween 20 only partially solubilises the precipitated enzyme under conditions which can precipitate and solubilise the human enzyme.

  17. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  18. 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

  19. 75 FR 66116 - Agency Information Collection Activities: Proposed Collection; Comment Request, OMB No. 1660-NEW...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-27

    ...; Comment Request, OMB No. 1660-NEW; Logistics Capability Assessment Tool (LCAT) AGENCY: Federal Emergency... Logistics Capability Assessment Tool. DATES: Comments must be submitted on or before December 27, 2010..., Logistics Management Directorate, Logistics Plans and Exercises Division, 202-212-7323 for...

  20. 78 FR 73872 - Agency Information Collection Activities: Proposed Collection; Comment Request; Logistics...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-09

    ... or material, all submissions will be posted, without change, to the Federal eRulemaking Portal at... further enhance strengths. The LCAT is authorized by Public Law 109-295, Department of Homeland Security... their disaster logistics planning and response capabilities and identify areas of relative strength...

  1. Effect of 17alpha-ethinylestradiol on activity of rat liver enzymes for synthesis and hydrolysis of cholesterol esters

    SciTech Connect

    Nikitin, Yu.P.; Dushkin, M.I.; Dolgov, A.V.; Gordienko, I.A.

    1987-01-01

    Administration of estrogens is known to lower the concentration of cholesterol esters in the blood vessel wall and may delay the development of arteriosclerosis. It is also known that under the influence of estrogens the redistribution of concentrations of free cholesterol and cholesterol esters takes place in rats between the blood and liver as a result of the intensification of receptor-dependent uptake of low-density lipoproteins by the hepatocytes. The mechanisms of this intracellular redistribution, however, have been inadequately studied. The purpose of this paper is to study the effects of 17alpha-ethinylestradiol on the activity of lysosomal and cytoplasmic cholesterol esterases, acyl-CoA-cholesterol-O-acyltransferase, lysosomal acid phosphatase, and beta-D-galactosidase. The activity was measured by using cholesterol (1-C 14)-oleate as the substrate. The influence of the estradiol is found to be based on cholesterol redistribution between the blood and liver. Accumulation of free cholesterol in the liver under these conditions stimulates bile acid formation. Depression of cholesterol ester synthesis as a result of direct inhibition of the acyltransferase by the estradiol is found to possibly contribute to the fall in the cholesterol level in the body. Liquid scintillation counting was used to measure distribution and accumulation.

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

    PubMed

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

    2004-08-01

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

  3. Effect of administration with the extract of Gymnema sylvestre R. Br leaves on lipid metabolism in rats.

    PubMed

    Shigematsu, N; Asano, R; Shimosaka, M; Okazaki, M

    2001-06-01

    Extract of Gymnema sylvestre R. Br leaves (GE) was orally administered once a day to rats fed a high fat diet or normal fat diet for 3 weeks to investigate its influence on lipid metabolism. As a result, GE did not influence body weight gain or feed intake in both diet groups during the experimental period. The apparent fat digestibility was significantly decreased by GE in both diet groups for the last 2 weeks of the experimental period, though not the apparent protein digestibility. In addition, the excretion of neutral sterols and acid steroids into feces was increased by GE in both diet groups. Furthermore, GE decreased the total cholesterol and triglyceride levels in serum. On the other hand, blood lecithin-cholesterol acyltransferase (LCAT) activity was increased by GE. Moreover, it was suggested that GE influenced cecal fermentation and that propionic acid and acetic acid contents in cecum were significantly increased by GE. Consequently, it was suggested that GE improved serum cholesterol and triglyceride levels through influence over a wide range of lipid metabolism in rats.

  4. APOA1 — EDRN Public Portal

    Cancer.gov

    APOA1, a secreted protein belonging to the apolipoprotein A1/A4/E family, participates in the reverse transport of cholesterol from tissues to the liver for excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for the lecithin cholesterol acyltransferase (LCAT). As part of the SPAP complex, APOA1 activates spermatozoa motility. This protein is a major protein of plasma HDL and is also found in chylomicrons. It is synthesized in the liver and small intestine. Defects in APOA1 are a cause of several diseases: 1) high density lipoprotein deficiency type 2 (HDLD2), also known as familial hypoalphalipoproteinemia, for which inheritance is autosomal dominant; 2) the low HDL levels observed in high density lipoprotein deficiency type 1 (HDLD1), also known as analphalipoproteinemia or Tangier disease (TGD), a recessive disorder characterized by the absence of plasma HDL, accumulation of cholesteryl esters, premature coronary artery disease, hepatosplenomegaly, recurrent peripheral neuropathy and progressive muscle wasting and weakness; 3) amyloid polyneuropathy-nephropathy Iowa type (AMYLIOWA), also known as amyloidosis van Allen type or familial amyloid polyneuropathy type III, a hereditary generalized amyloidosis due to deposition of amyloid mainly constituted by apolipoprotein A1; 4) amyloidosis type 8 (AMYL8), also known as systemic non-neuropathic amyloidosis or Ostertag-type amyloidosis, a hereditary generalized amyloidosis due to deposition of apolipoprotein A1, fibrinogen and lysozyme amyloids.

  5. Biosynthesis of gallotannins : Enzymatic 'disproportionation' of 1,6-digalloylglucose to 1,2,6-trigalloylglucose and 6-galloylglucose by an acyltransferase from leaves of Rhus typhina L.

    PubMed

    Denzel, K; Gross, G G

    1991-05-01

    Cell-free extracts from leaves of Rhus typhina L. (sumach) were found to transfer the 1-O-galloyl moiety of l,6-di-O-galloyl-β-D-glucose to the 2-position of the same compound, yielding 1,2,6-tri-O-galloyl-β-D-glucose and leaving 6-O-galloylglucose as the deacylated by-product. The enzyme catalyzing this 'disproportionation' was purified almost 1700-fold. It had a molecular weight of approx. 56 000, a K m value of 11.5 mM, was stable between pH 4.5 and 6.5, and most active at pH 5.9 and 40° C. The systematic name "1,6-di-O-galloyl-glucose: 1,6-di-O-galloylglucose 2-O-galloyltransferase" (EC 2.3.1.) was proposed for this new enzyme whose detection provided evidence that, in addition to β-glucogallin (1-O-galloyl-β-D-glucose), higher substituted glucose esters also have the potential to serve as acyl donors in the biosynthesis of gallotannins.

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

    PubMed

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

    2007-07-03

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

  7. Deficiency of serum cholesteryl-ester transfer activity in patients with familial hyperalphalipoproteinaemia.

    PubMed

    Koizumi, J; Mabuchi, H; Yoshimura, A; Michishita, I; Takeda, M; Itoh, H; Sakai, Y; Sakai, T; Ueda, K; Takeda, R

    1985-12-01

    Lipoprotein patterns and cholesteryl ester transfer activity (CETA) were examined in 2 patients with familial hyperalphalipoproteinaemia (FHALP). The proband was a healthy 58-year-old Japanese male who had an HDL cholesterol of 7.83 mmol/l (301 mg/dl). His sister's HDL cholesterol was 4.52 mmol/l (174 mg/dl), which suggested that both were homozygous carriers of FHALP. In both subjects HDL showed a high cholesterol/apo A-I ratio and appeared to be a larger-sized particle than normal HDL on agarose gel chromatography. Two of the proband's children showed higher HDL cholesterol levels (1.74 mmol/l, 2.16 mmol/l) than normal, but another 2 children showed normal levels (1.48 mmol/l, 1.40 mmol/l). However, the ratios of HDL cholesterol to total cholesterol and to apo A-I in all children were higher than normal. These data suggest, but do not prove, that all his children were heterozygotes. Apo B levels in all of the family members studied were lower than normal (47-80 mg/dl). Deceased members of the same family had not died from cardiovascular disease. Cholesteryl-ester transfer activity was studied in both patients. When serum or lipoprotein deficient serum (d greater than 1.21) and [3H]cholesteryl ester labelled HDL3 were incubated in the presence of an LCAT inhibitor, there was no evidence of cholesteryl ester transfer from HDL to VLDL and/or LDL, unlike normal subjects. The deficiency of CETA in these patients with FHALP presumably accounted for the increase in particle size and cholesterol enrichment of HDL.

  8. Detection of 1-O-malylglucose: pelargonidin 3-O-glucose-6''-O-malyltransferase activity in carnation (Dianthus caryophyllus).

    PubMed

    Abe, Yutaka; Tera, Masayuki; Sasaki, Nobuhiro; Okamura, Masachika; Umemoto, Naoyuki; Momose, Masaki; Kawahara, Nobuo; Kamakura, Hiroyuki; Goda, Yukihiro; Nagasawa, Kazuo; Ozeki, Yoshihiro

    2008-09-05

    Carnations have anthocyanins acylated with malate. Although anthocyanin acyltransferases have been reported in several plant species, anthocyanin malyltransferase (AMalT) activity in carnation has not been identified. Here, an acyl donor substance of AMalT, 1-O-beta-D-malylglucose, was extracted and partially purified from the petals of carnation. This was synthesized chemically to analyze AMalT activity in a crude extract from carnation. Changes in the AMalT activity showed close correlation to the accumulation of pelargonidin 3-malylglucoside (Pel 3-malGlc) during the development of red petals of carnation, but neither AMalT activity nor Pel 3-malGlc accumulation was detectable in roots, stems and leaves.

  9. Overexpression of a BAHD Acyltransferase, OsAt10, Alters Rice Cell Wall Hydroxycinnamic Acid Content and Saccharification1[C][W][OA

    PubMed Central

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

    2013-01-01

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

  10. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

    PubMed

    Rios-Esteves, Jessica; Resh, Marilyn D

    2013-09-26

    Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

  11. Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis*

    PubMed Central

    Nigam, Aeshna; Almabruk, Khaled H.; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N.; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-01-01

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains. PMID:24923585

  12. Modification of rifamycin polyketide backbone leads to improved drug activity against rifampicin-resistant Mycobacterium tuberculosis.

    PubMed

    Nigam, Aeshna; Almabruk, Khaled H; Saxena, Anjali; Yang, Jongtae; Mukherjee, Udita; Kaur, Hardeep; Kohli, Puneet; Kumari, Rashmi; Singh, Priya; Zakharov, Lev N; Singh, Yogendra; Mahmud, Taifo; Lal, Rup

    2014-07-25

    Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.

  13. IL-1. alpha. increases arachidonyl-CoA: Lysophospholipid acyltransterase activity and stimulates ( sup 3 H) arachidonate incorporation into phospholipids in rat mesangial cells

    SciTech Connect

    Nakazato, Y.; Sedor, J.R. )

    1992-01-01

    The proinflammatory cytokine interleukin-1{alpha} is a potent stimulus of prostaglandin synthesis. The authors have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A{sub 2}. Phospholipase A{sub 2}. Phospholipase A{sub 2} activation results in the formation of lysophospholipids and free fatty acids. They now investigate the effects of IL-1{alpha} on reacylation of lysophospholipids. Incubations with IL-1{alpha} for 24 hours significantly stimulated mesangial cell ({sup 3}H)arachidonic acid incorporation but not ({sup 3}H)oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. They conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.

  14. Enhancement of Local Climate Analysis Tool

    NASA Astrophysics Data System (ADS)

    Horsfall, F. M.; Timofeyeva, M. M.; Dutton, J.

    2012-12-01

    The National Oceanographic and Atmospheric Administration (NOAA) National Weather Service (NWS) will enhance its Local Climate Analysis Tool (LCAT) to incorporate specific capabilities to meet the needs of various users including energy, health, and other communities. LCAT is an online interactive tool that provides quick and easy access to climate data and allows users to conduct analyses at the local level such as time series analysis, trend analysis, compositing, correlation and regression techniques, with others to be incorporated as needed. LCAT uses principles of Artificial Intelligence in connecting human and computer perceptions on application of data and scientific techniques in multiprocessing simultaneous users' tasks. Future development includes expanding the type of data currently imported by LCAT (historical data at stations and climate divisions) to gridded reanalysis and General Circulation Model (GCM) data, which are available on global grids and thus will allow for climate studies to be conducted at international locations. We will describe ongoing activities to incorporate NOAA Climate Forecast System (CFS) reanalysis data (CFSR), NOAA model output data, including output from the National Multi Model Ensemble Prediction System (NMME) and longer term projection models, and plans to integrate LCAT into the Earth System Grid Federation (ESGF) and its protocols for accessing model output and observational data to ensure there is no redundancy in development of tools that facilitate scientific advancements and use of climate model information in applications. Validation and inter-comparison of forecast models will be included as part of the enhancement to LCAT. To ensure sustained development, we will investigate options for open sourcing LCAT development, in particular, through the University Corporation for Atmospheric Research (UCAR).

  15. ACAT inhibitory activity of exudates from Calocedrus macrolepis var. formosana.

    PubMed

    Hsieh, Yu-Hsin; Chen, Kuan-Jung; Chien, Shih-Chang; Cheng, Wen-Ling; Xiao, Jun-Hong; Wang, Sheng-Yang

    2012-12-01

    Cholesterol acyltransferase (ACAT) is an enzyme controlling cholesterol esterification in cells. Large amounts of cholesterol esters accumulate in macrophages and smooth muscle cells of blood vessel walls resulting in the initial stages of atherosclerosis. Thus, atherosclerosis might be inhibited through inhibition of the activity of ACAT. In the present study, we identified by spectral analysis and chromatographic quantification that ferruginol was the most abundant component of exudates secreted from the wounding site of Calocedrus macrolepis Kurz var. formosana. Results obtained from the cholesterol absorption assay revealed that ferruginol exhibited a significant inhibitory activity on cholesterol absorption in mice macrophages (RAW 264.7 cell). Based on the results from analyzing the ratio of cholesterol esterification, ferruginol dose-dependently suppressed cholesterol esterification and the IC50 value was 2.0 microg/mL. In conclusion, ferruginol revealed strong inhibitory activities that retarded the absorption and esterification of cholesterol in cells. Our finding indicates that ferruginol might possess a potential for development as a pharmaceutical product for preventing arteriosclerosis.

  16. Different Functional and Structural Characteristics between ApoA-I and ApoA-4 in Lipid-Free and Reconstituted HDL State: ApoA-4 Showed Less Anti-Atherogenic Activity

    PubMed Central

    Yoo, Jeong-Ah; Lee, Eun-Young; Park, Ji Yoon; Lee, Seung-Taek; Ham, Sihyun; Cho, Kyung-Hyun

    2015-01-01

    Apolipoprotein A-I and A-IV are protein constituents of high-density lipoproteins although their functional difference in lipoprotein metabolism is still unclear. To compare anti-atherogenic properties between apoA-I and apoA-4, we characterized both proteins in lipid-free and lipid-bound state. In lipid-free state, apoA4 showed two distinct bands, around 78 and 67 Å on native gel electrophoresis, while apoA-I showed scattered band pattern less than 71 Å. In reconstituted HDL (rHDL) state, apoA-4 showed three major bands around 101 Å and 113 Å, while apoA-I-rHDL showed almost single band around 98 Å size. Lipid-free apoA-I showed 2.9-fold higher phospholipid binding ability than apoA-4. In lipid-free state, BS3-crosslinking revealed that apoA-4 showed less multimerization tendency upto dimer, while apoA-I showed pentamerization. In rHDL state (95:1), apoA-4 was existed as dimer as like as apoA-I. With higher phospholipid content (255:1), five apoA-I and three apoA-4 were required to the bigger rHDL formation. Regardless of particle size, apoA-I-rHDL showed superior LCAT activation ability than apoA-4-rHDL. Uptake of acetylated LDL was inhibited by apoA-I in both lipid-free and lipid-bound state, while apoA-4 inhibited it only lipid-free state. ApoA-4 showed less anti-atherogenic activity with more sensitivity to glycation. In conclusion, apoA-4 showed inferior physiological functions in lipid-bound state, compared with those of apoA-I, to induce more pro-atherosclerotic properties. PMID:25997739

  17. Luminol electrochemiluminescence for the analysis of active cholesterol at the plasma membrane in single mammalian cells.

    PubMed

    Ma, Guangzhong; Zhou, Junyu; Tian, Chunxiu; Jiang, Dechen; Fang, Danjun; Chen, Hongyuan

    2013-04-16

    A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.

  18. Cholesterol overloading leads to hepatic L02 cell damage through activation of the unfolded protein response.

    PubMed

    Li, Qi; Liu, Zhiguo; Guo, Jianli; Chen, Jiangyuan; Yang, Pu; Tian, Jun; Sun, Jun; Zong, Yiqiang; Qu, Shen

    2009-10-01

    Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.

  19. Effect of 360His mutation in apolipoprotein A-IV on plasma HDL-cholesterol response to dietary fat.

    PubMed

    Jansen, S; Lopez-Miranda, J; Ordovas, J M; Zambrana, J L; Marin, C; Ostos, M A; Castro, P; McPherson, R; Lopez Segura, F; Blanco, A; Jimenez Pereperez, J A; Perez-Jimenez, F

    1997-10-01

    In order to determine whether genetic variability of apolipoprotein (apo) A-IV is responsible for the improvement in lipid profile when dietary saturated fats are replaced by carbohydrates or monounsaturated fats, 41 healthy male subjects were studied: 33 were homozygous for the 360Gln allele and 8 were heterozygote carriers of the 360His allele. These were administered three consecutive 4-week diets. The first was a diet rich in saturated fat (SAT diet, with 38% fat, 20% saturated. This was followed by a low fat diet (NCEP-I, with < 30% fat, < 10% saturated). The final diet was rich in monounsaturated fat (MUFA diet, with 38% fat, 22% monounsaturated). There was no difference in plasma lipid and apolipoprotein levels of both groups of individuals after consuming the SAT diet. Switching from this diet to the NCEP-I diet, carriers of the 360His allele presented a greater decrease in high density lipoprotein-cholesterol (HDL-C) (-10 vs. -1 mg/dL, P < 0.004) and apoA-I levels (-19 vs. -8 mg/dL, P < 0.037). Similarly, replacement of carbohydrates by monounsaturated fats produced a greater increase in HDL-C (9 vs. 1 mg/dL, P < 0.003) and apoA-I levels (9 vs. 2 mg/dL, P < 0.036) in carriers of the 360His mutation. Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) activities and apoA-IV levels were also measured. However, no genotype-related differences were observed for these parameters. Our results suggest that variability in HDL-C and apoA-I response to diet is, at least partially, determined by the 360His mutation of apoA-IV.

  20. Volatile emission after controlled atmosphere storage of Mondial Gala apples (Malus domestica): relationship to some involved enzyme activities.

    PubMed

    Lara, Isabel; Echeverría, Gemma; Graell, Jordi; López, María Luisa

    2007-07-25

    Mondial Gala apples were harvested at commercial maturity and stored at 1 degrees C under either air or controlled atmosphere (CA) conditions (2 kPa O2/2 kPa CO2 and 1 kPa O2/1 kPa CO2), where they remained for 3 or 6 months. Data on emission of selected volatile esters, alcohol precursors, and activity of some aroma-related enzymes in both peel and pulp tissues were obtained during subsequent shelf life of fruit and submitted to multivariate analysis procedures. CA storage caused a decrease in the emission of volatile esters in comparison to storage in air. Results suggest that lessened ester production was the consequence of modifications in activities of alcohol o-acyltransferase (AAT) and lipoxygenase (LOX) activities. For short-term storage, inhibition of lipoxygenase activity in CA stored fruit possibly led to a shortage of lipid-derived substrates, resulting in decreased production of volatile esters in spite of substantial ester-forming capacity that allowed for some recovery of fruit capacity for ester emission during the shelf life. For long-term storage, strong inhibition of AAT activity in CA stored fruit in combination with low LOX activities resulted in unrecoverable diminution of biosynthesis of volatile esters.

  1. Absolute stereochemistry of fungal beauveriolide III and ACAT inhibitory activity of four stereoisomers.

    PubMed

    Ohshiro, Taichi; Namatame, Ichiji; Nagai, Kenichiro; Sekiguchi, Takafumi; Doi, Takayuki; Takahashi, Takashi; Akasaka, Kazuaki; Rudel, Lawrence L; Tomoda, Hiroshi; Omura, Satoshi

    2006-09-29

    Fungal beauveriolide III (BeauIII, 1b), a cyclodepsipeptide inhibiting acyl-CoA:cholesterol acyltransferase (ACAT) and showing antiatherogenic activity in mouse models, consists of L-Phe, L-Ala, D-allo-Ile, and 3-hydroxy-4-methyloctanoic acid (HMA) moieties, but the stereochemistry of the HMA part has not until now been fully defined. To determine it, four HMA stereoisomers were synthesized and labeled with (S)-(+)-2-(anthracene-2,3-dicarboximido)-1-propyl trifluoromethane sulfonate (AP-OTf), a chiral fluorescent reagent. The derivatives were separated by HPLC and compared with the natural HMA derivative, which was thereby identified as (3S,4S)HMA in BeauIII. Furthermore, the four beauveriolide III isomers ((3S,4S)BeauIII (23a), (3R,4R)BeauIII (23b), (3R,4S)BeauIII (23c), and (3S,4R)BeauIII (23d)) were synthesized, and it was shown that all the spectral data for 23a were identical with those for natural 1b. Isomers 23a and 23d showed potent inhibitory activity of lipid droplet accumulation in macrophages, while the other two isomers caused weak inhibition. Thus, the 3S configuration of BeauIII is important for this activity. Furthermore, 23a and 23d showed rather specific inhibition against the ACAT1 isozyme.

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

    PubMed

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

    2011-01-01

    Hawthorn (Crataegus pinnatifida) is an edible fruit used in traditional Chinese medicine to lower plasma lipids. This study explored lipid-lowering compounds and underlying mechanisms of action of hawthorn. Hawthorn powder extracts inhibited acylCoA:cholesterol acyltransferase (ACAT) activity in Caco-2 cells. The inhibitory activity was positively associated with triterpenic acid (i.e., oleanolic acid (OA) and ursolic acid (UA)) contents in the extracts. Cholesterol lowering effects of hawthorn and its potential additive effect in combination with plant sterol esters (PSE) were further studied in hamsters. Animals were fed a semi-synthetic diet containing 0.08% (w/w) cholesterol (control) or the same diet supplemented with (i) 0.37% hawthorn dichloromethane extract, (ii) 0.24% PSE, (iii) hawthorn dichloromethane extract (0.37%) plus PSE (0.24%) or (iv) OA/UA mixture (0.01%) for 4 weeks. Compared to the control diet, hawthorn, PSE, hawthorn plus PSE and OA/UA significantly lowered plasma non-HDL (VLDL + LDL) cholesterol concentrations by 8%, 9%, 21% and 6% and decreased hepatic cholesterol ester content by 9%, 23%, 46% and 22%, respectively. The cholesterol lowering effects of these ingredients were conversely associated with their capacities in increasing fecal neutral sterol excretion. In conclusion, OA and UA are responsible for the cholesterol lowering effect of hawthorn by inhibiting intestinal ACAT activity. In addition, hawthorn and particularly its bioactive compounds (OA and UA) enhanced the cholesterol lowering effect of plant sterols.

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

    PubMed

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

    2012-10-25

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

  4. In vitro metabolism of pyripyropene A and ACAT inhibitory activity of its metabolites.

    PubMed

    Matsuda, Daisuke; Ohshiro, Taichi; Ohtawa, Masaki; Yamazaki, Hiroyuki; Nagamitsu, Tohru; Tomoda, Hiroshi

    2015-01-01

    Pyripyropene A (PPPA, 1) of fungal origin, a selective inhibitor of acyl-CoA:cholesterol acyltransferase 2 (ACAT2), proved orally active in atherogenic mouse models. The in vitro metabolites of 1 in liver microsomes and plasma of human, rabbit, rat and mouse were analyzed by ultra fast liquid chromatography and liquid chromatography/tandem mass spectrometry. In the liver microsomes from all species, successive hydrolysis occurred at the 1-O-acetyl residue, then at the 11-O-acetyl residue of 1, while the 7-O-acetyl residue was resistant to hydrolysis. Furthermore, dehydrogenation of the newly generated 11-alcoholic hydroxyl residue occurred in human and mouse-liver microsomes, while oxidation of the pyridine ring occurred in human and rabbit liver microsomes. On the other hand, hydrolysis of the 7-O-acetyl residue proceeded only in the mouse plasma. These data indicated that the in vitro metabolic profiles of 1 have subtle differences among animal species. All of the PPPA metabolites observed in liver microsomes and plasma markedly decreased ACAT2 inhibitory activity. These findings will help us to synthesize new PPPA derivatives more effective in in vivo study than 1.

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

    PubMed

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

    2001-03-30

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

  6. Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

    SciTech Connect

    Pols, Thijs W.H.; Ottenhoff, Roelof; Vos, Mariska; Levels, Johannes H.M.; Quax, Paul H.A.; Meijers, Joost C.M.; Pannekoek, Hans; Groen, Albert K.; Vries, Carlie J.M. de

    2008-02-22

    NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor, and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity.

  7. Effect of various eicosanoid products of arachidonic acid on the acyl CoA: Cholesterol acyl transferase activity in three different mammalian cell lines

    SciTech Connect

    Malo, P.El.

    1988-01-01

    Acylcoenzyme A:cholesterol acyltransferase (ACAT) catalyzes cholesterol ester synthesis intracellularly and has been implicated in the development of atherosclerosis. An in vitro assay has been adapted for determining ACAT activity from rat FU5AH hepatoma, Chinese hamster ovary (CHO) and rat thoracic aortic smooth muscle (RSM) cells. Formation of {sup 14}C-labelled cholesteryl oleate at 0 to 60 min {plus minus} cholesterol was determined; in the presence of exogenous cholesterol, ACAT activity was approximately linear and surpassed the plateau observed in ACAT activity without cholesterol. Increasing exogenous cholesterol concentration, the amount of oleoyl CoA or the amount of microsomal protein produced a corresponding increase in ACAT activity, while ester formation was slightly increased by decreasing the ratio of Triton WR-1339 to cholesterol. Both the thromboxane A{sub 2} (TxA{sub 2}) mimic, U-44069, and the inflammatory lipoxygenase product, LTB{sub 4}, decreased optimal in vitro microsomal ACAT activity from RSM, but not form FU5AH, while CHO ACAT activity was suppressed by LTB{sub r} only. PGI{sub 2}, PGE{sub 2} and PGF{sub 2{alpha}} had minimal effects for each cell type.

  8. Rho2 Palmitoylation Is Required for Plasma Membrane Localization and Proper Signaling to the Fission Yeast Cell Integrity Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Sánchez-Mir, Laura; Franco, Alejandro; Martín-García, Rebeca; Madrid, Marisa; Vicente-Soler, Jero; Soto, Teresa; Gacto, Mariano; Pérez, Pilar

    2014-01-01

    The fission yeast small GTPase Rho2 regulates morphogenesis and is an upstream activator of the cell integrity pathway, whose key element, mitogen-activated protein kinase (MAPK) Pmk1, becomes activated by multiple environmental stimuli and controls several cellular functions. Here we demonstrate that farnesylated Rho2 becomes palmitoylated in vivo at cysteine-196 within its carboxyl end and that this modification allows its specific targeting to the plasma membrane. Unlike that of other palmitoylated and prenylated GTPases, the Rho2 control of morphogenesis and Pmk1 activity is strictly dependent upon plasma membrane localization and is not found in other cellular membranes. Indeed, artificial plasma membrane targeting bypassed the Rho2 need for palmitoylation in order to signal. Detailed functional analysis of Rho2 chimeras fused to the carboxyl end from the essential GTPase Rho1 showed that GTPase palmitoylation is partially dependent on the prenylation context and confirmed that Rho2 signaling is independent of Rho GTP dissociation inhibitor (GDI) function. We further demonstrate that Rho2 is an in vivo substrate for DHHC family acyltransferase Erf2 palmitoyltransferase. Remarkably, Rho3, another Erf2 target, negatively regulates Pmk1 activity in a Rho2-independent fashion, thus revealing the existence of cross talk whereby both GTPases antagonistically modulate the activity of this MAPK cascade. PMID:24820419

  9. The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

    PubMed

    Gorné, Lucas D; Acosta-Rodríguez, Victoria A; Pasquaré, Susana J; Salvador, Gabriela A; Giusto, Norma M; Guido, Mario Eduardo

    2015-02-01

    The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

  10. Identification of loci of Pseudomonas syringae pv. actinidiae involved in lipolytic activity and their role in colonization of kiwifruit leaves.

    PubMed

    Kumar Patel, Hitendra; Ferrante, Patrizia; Xianfa, Meng; Javvadi, Sree Gowrinadh; Subramoni, Sujatha; Scortichini, Marco; Venturi, Vittorio

    2017-01-23

    Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa), an emerging pathogen of kiwifruit plants, has recently brought about major economic losses worldwide. Genetic studies on virulence functions of Psa have not yet been reported and there is little experimental data regarding bacterial genes involved in pathogenesis. In this study, we performed a genetic screen in order to identify transposon mutants altered in the lipolytic activity as it is known that mechanisms of regulation, production and secretion of enzymes often play crucial roles in virulence of plant pathogens. We aimed to identify the set of secretion and global regulatory loci that control lipolytic activity and also play important roles in in planta fitness. Our screen for altered lipolytic activity phenotype identified a total of 58 Tn5 transposon mutants. Mapping all these Tn5 mutants revealed that the transposons were inserted in genes that play roles in cell division, chemotaxis, metabolism, movement, recombination, regulation, signal transduction, and transport as well as a few unknown functions. Several of these identified Psa Tn5 mutants, notably the functions affected in phosphomannomutase AlgC, lipid A biosynthesis acyltransferase, glutamate--cysteine ligase and the type IV pilus protein PilI, were also found affected in in planta survival and/or growth in kiwifruit plants. The results of the genetic screen and identification of novel loci involved in in planta fitness of Psa are presented and discussed.

  11. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    DTIC Science & Technology

    2015-10-01

    accumulation. Subsequently, nuclear YAP/TAZ binds to TEA /TEF (transcriptional enhancer factor)-domain tran- scription factors (TEAD1–TEAD4 in mammals and...Xu Wu1* TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway...approaches reveal that TEA domain (TEAD) transcription factors are palmitoylated. (a) Structures of the chemical reporter of palmitoylation, 1, and the

  12. A vernonia diacylglycerol acyltransferase can increase renewable oil production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increasing the production of plant oils such as soybean oil, a critical renewable resource for food and fuel, will be highly valuable. Successful breeding for higher oil levels in soybean, however, usually results in reduced protein, a second valuable seed component. We show that by manipulating a h...

  13. Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

    DTIC Science & Technology

    2014-08-01

    which were previously unrecognized as PATs, might catalyze protein palmitoylation. For example, lysophosphatidylcholine acyltrans- ferase 1 (Lpcat1...R. K. (2011) Acyl-CoA:lysophosphatidylcholine acyl- transferase I (Lpcat1) catalyzes histone protein O-palmitoylation to regulate mRNA synthesis. J...Chem. 42, 4932−4941. (30) Mani, N. S., and Townsend, C. A. (1997) A concise synthesis of (+)-cerulenin from a chiral oxiranyllithium. J. Org. Chem. 62

  14. Dihydrolipoamide acyltransferase is critical for Mycobacterium tuberculosis pathogenesis.

    PubMed

    Shi, Shuangping; Ehrt, Sabine

    2006-01-01

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

  15. A novel technical approach for the measurement of individual ACAT-1 and ACAT-2 enzymatic activity in the testis.

    PubMed

    Chen, Li; Lafond, Julie; Pelletier, R-Marc

    2009-01-01

    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC(50) values of 100 and 1,000 microM, respectively, in the tubules. Next, the inhibitor concentration (100 microM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 microg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 microg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0-1,000 microM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.

  16. Corn silk extract improves cholesterol metabolism in C57BL/6J mouse fed high-fat diets

    PubMed Central

    Cha, Jae Hoon; Kim, Sun Rim; Kang, Hyun Joong; Kim, Myung Hwan; Ha, Ae Wha

    2016-01-01

    BACKGROUND/OBJECTIVES Corn silk (CS) extract contains large amounts of maysin, which is a major flavonoid in CS. However, studies regarding the effect of CS extract on cholesterol metabolism is limited. Therefore, the purpose of this study was to determine the effect of CS extract on cholesterol metabolism in C57BL/6J mouse fed high-fat diets. MATERIALS/METHODS Normal-fat group fed 7% fat diet, high-fat (HF) group fed 25% fat diet, and high-fat with corn silk (HFCS) group were orally administered CS extract (100 mg/kg body weight) daily. Serum and hepatic levels of total lipids, triglycerides, and total cholesterol as well as serum free fatty acid, glucose, and insulin levels were determined. The mRNA expression levels of acyl-CoA: cholesterol acyltransferase (ACAT), cholesterol 7-alpha hydroxylase (CYP7A1), farnesoid X receptor (FXR), lecithin cholesterol acyltransferase (LCAT), low-density lipoprotein receptor, 3-hyroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), adiponectin, leptin, and tumor necrosis factor α were determined. RESULTS Oral administration of CS extract with HF improved serum glucose and insulin levels as well as attenuated HF-induced fatty liver. CS extracts significantly elevated mRNA expression levels of adipocytokines and reduced mRNA expression levels of HMG-CoA reductase, ACAT, and FXR. The mRNA expression levels of CYP7A1 and LCAT between the HF group and HFCS group were not statistically different. CONCLUSIONS CS extract supplementation with a high-fat diet improves levels of adipocytokine secretion and glucose homeostasis. CS extract is also effective in decreasing the regulatory pool of hepatic cholesterol, in line with decreased blood and hepatic levels of cholesterol though modulation of mRNA expression levels of HMG-CoA reductase, ACAT, and FXR. PMID:27698957

  17. Anti-melanoma activity of Forsythiae Fructus aqueous extract in mice involves regulation of glycerophospholipid metabolisms by UPLC/Q-TOF MS-based metabolomics study

    PubMed Central

    Bao, Jiaolin; Liu, Fang; Zhang, Chao; Wang, Kai; Jia, Xuejing; Wang, Xiaotong; Chen, Meiwan; Li, Peng; Su, Huanxing; Wang, Yitao; Wan, Jian-Bo; He, Chengwei

    2016-01-01

    Metabolomics is a comprehensive assessment of endogenous metabolites of a biological system in a holistic context. In this study, we evaluated the in vivo anti-melanoma activity of aqueous extract of Forsythiae Fructus (FAE) and globally explored the serum metabolome characteristics of B16-F10 melanoma-bearing mice. UPLC/Q-TOF MS combined with pattern recognition approaches were employed to examine the comprehensive metabolic signatures and differentiating metabolites. The results demonstrated that FAE exhibited remarkable antitumor activity against B16-F10 melanoma in C57BL/6 mice and restored the disturbed metabolic profile by tumor insult. We identified 17 metabolites which were correlated with the antitumor effect of FAE. Most of these metabolites are involved in glycerophospholipid metabolisms. Notably, several lysophosphatidylcholines (LysoPCs) significantly decreased in tumor model group, while FAE treatment restored the changes of these phospholipids to about normal condition. Moreover, we found that lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX) were highly expressed in melanoma, and FAE markedly down-regulated their expression. These findings indicated that modulation of glycerophospholipid metabolisms may play a pivotal role in the growth of melanoma and the antitumor activity of FAE. Besides, our results suggested that serum LysoPCs could be potential biomarkers for the diagnosis and prognosis of melanoma and other malignant tumors. PMID:27991567

  18. Delta 6- and delta 12-desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis).

    PubMed Central

    Griffiths, G; Stobart, A K; Stymne, S

    1988-01-01

    Microsomal membrane preparations from the maturing cotyledons of common borage (Borago officinalis) exhibit delta 12- and delta 6-desaturase activities, which resulted in the synthesis of linoleate and gamma-linolenate respectively. The desaturase enzymes utilized the complex lipid substrate phosphatidylcholine. The activity of these enzymes was sufficiently high to allow the monitoring of the mass changes in the endogenous oleate, linoleate and gamma-linolenate in the microsomal phosphatidylcholine in the presence of NADH (i.e. under desaturating conditions). The results illustrate that the delta 12-desaturase uses the oleate substrate at both the sn-1 and -2 positions of sn-phosphatidylcholine, whereas the delta 6-desaturase is almost totally restricted to the linoleate at position 2 of the complex lipid. Estimate of the acyl-substrate pool size at position 2 of sn-phosphatidylcholine for both desaturases indicated that some 50% of the oleate and linoleate was available to the enzymes. The microsomes (microsomal fractions) had a somewhat impaired Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway for the formation of triacylglycerols when compared with other oil-rich plant species that have been studied [Stymne & Stobart (1987) The Biochemistry of Plants: a Comprehensive Treatise (Stumpf, P.K., ed.), vol. 10, chapter 8, pp. 175-214, Academic Press, New York]. In the presence of sn-glycerol 3-phosphate and acyl-CoA, large quantities of phosphatidic acid accumulated in the membranes. Acyl-selectivity studies on the glycerol-acylating enzymes showed that gamma-linolenate could be acylated to both the sn-1 and sn-2 positions of sn-glycerol 3-phosphate. However, stereochemical analysis of the acyl components of the sn-triacylglycerol obtained from mature seeds indicated that, whereas no gamma-linolenate was present at the sn-1 position, it accounted for over 50% of the fatty acids at position sn-3. The results indicate that the diacylglycerol

  19. Delta 6- and delta 12-desaturase activities and phosphatidic acid formation in microsomal preparations from the developing cotyledons of common borage (Borago officinalis).

    PubMed

    Griffiths, G; Stobart, A K; Stymne, S

    1988-06-15

    Microsomal membrane preparations from the maturing cotyledons of common borage (Borago officinalis) exhibit delta 12- and delta 6-desaturase activities, which resulted in the synthesis of linoleate and gamma-linolenate respectively. The desaturase enzymes utilized the complex lipid substrate phosphatidylcholine. The activity of these enzymes was sufficiently high to allow the monitoring of the mass changes in the endogenous oleate, linoleate and gamma-linolenate in the microsomal phosphatidylcholine in the presence of NADH (i.e. under desaturating conditions). The results illustrate that the delta 12-desaturase uses the oleate substrate at both the sn-1 and -2 positions of sn-phosphatidylcholine, whereas the delta 6-desaturase is almost totally restricted to the linoleate at position 2 of the complex lipid. Estimate of the acyl-substrate pool size at position 2 of sn-phosphatidylcholine for both desaturases indicated that some 50% of the oleate and linoleate was available to the enzymes. The microsomes (microsomal fractions) had a somewhat impaired Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway for the formation of triacylglycerols when compared with other oil-rich plant species that have been studied [Stymne & Stobart (1987) The Biochemistry of Plants: a Comprehensive Treatise (Stumpf, P.K., ed.), vol. 10, chapter 8, pp. 175-214, Academic Press, New York]. In the presence of sn-glycerol 3-phosphate and acyl-CoA, large quantities of phosphatidic acid accumulated in the membranes. Acyl-selectivity studies on the glycerol-acylating enzymes showed that gamma-linolenate could be acylated to both the sn-1 and sn-2 positions of sn-glycerol 3-phosphate. However, stereochemical analysis of the acyl components of the sn-triacylglycerol obtained from mature seeds indicated that, whereas no gamma-linolenate was present at the sn-1 position, it accounted for over 50% of the fatty acids at position sn-3. The results indicate that the diacylglycerol

  20. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

    PubMed Central

    Fernández-Valverde, M; Reglero, A; Martinez-Blanco, H; Luengo, J M

    1993-01-01

    Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. Images PMID:8476289

  1. Activation of PPARalpha and PPARgamma reduces triacylglycerol synthesis in rat hepatoma cells by reduction of nuclear SREBP-1.

    PubMed

    König, Bettina; Koch, Alexander; Spielmann, Julia; Hilgenfeld, Christian; Hirche, Frank; Stangl, Gabriele I; Eder, Klaus

    2009-03-01

    Fibrates and thiazolidinediones, agonists of PPARalpha and PPARgamma, respectively, reduce triglyceride concentrations in rat liver and plasma. Fatty acid and triacylglycerol synthesis in mammals is regulated by sterol regulatory element-binding protein (SREBP)-1c. Recently, it was shown that insulin-induced gene (Insig)-1, the key regulator of SREBP activity, is up-regulated by both activation of PPARalpha and PPARgamma. In order to elucidate whether inhibition of SREBP-1 activation may contribute to the triacylglycerol lowering effect of PPARalpha and PPARgamma agonists, we incubated rat hepatoma Fao cells with WY 14,643 and troglitazone, strong and selective agonists of PPARalpha and PPARgamma, respectively. Activation of both, PPARalpha and PPARgamma led to increased concentrations of Insig-1 and Insig-2a, with the most prominent effect on Insig-2a after troglitazone incubation. As a result, the amount of nuclear SREBP-1 was reduced in Fao cells by both WY 14,643 and troglitazone treatment. The reduction of nuclear SREBP-1 was associated with decreased mRNA concentrations of its target genes fatty acid synthase and glycerol-3-phosphate acyltransferase, implicated in fatty acid and triacylglycerol synthesis. This was finally reflected in reduced rates of newly synthesized triacylglycerols from de novo-derived fatty acids and decreased intracellular and secreted triacylglycerol concentrations in Fao cells treated with WY 14,643 and troglitazone, respectively. Thus, these data suggest that the triacylglycerol reducing effect of fibrates and thiazolidinediones is partially caused by inhibition of SREBP-1 activation via up-regulation of Insig.

  2. Cholesterol-Lowering Activity of Tartary Buckwheat Protein.

    PubMed

    Zhang, Chengnan; Zhang, Rui; Li, Yuk Man; Liang, Ning; Zhao, Yimin; Zhu, Hanyue; He, Zouyan; Liu, Jianhui; Hao, Wangjun; Jiao, Rui; Ma, Ka Ying; Chen, Zhen-Yu

    2017-03-08

    Previous research has shown that Tartary buckwheat flour is capable of reducing plasma cholesterol. The present study was to examine the effect of rutin and Tartary buckwheat protein on plasma total cholesterol (TC) in hypercholesterolemia hamsters. In the first animal experiment, 40 male hamsters were divided into four groups fed either the control diet or one of the three experimental diets containing 8.2 mmol rutin, 8.2 mmol quercetin, or 2.5 g kg(-1) cholestyramine, respectively. Results showed that only cholestyramine but not rutin and its aglycone quercetin decreased plasma TC, which suggested that rutin was not the active ingredient responsible for plasma TC-lowering activity of Tartary buckwheat flour. In the second animal experiment, 45 male hamsters were divided into five groups fed either the control diet or one of the four experimental diets containing 24% Tartary buckwheat protein, 24% rice protein, 24% wheat protein, or 5 g kg(-1) cholestyramine, respectively. Tartary buckwheat protein reduced plasma TC more effectively than cholestyramine (45% versus 37%), while rice and wheat proteins only reduced plasma TC by 10-13%. Tartary buckwheat protein caused 108% increase in the fecal excretion of total neutral sterols and 263% increase in the fecal excretion of total acidic sterols. real-time polymerase chain reaction and Western blotting analyses showed that Tartary buckwheat protein affected the gene expression of intestinal Niemann-Pick C1-like protein 1 (NPC1L1), acyl CoA:cholesterol acyltransferase 2 (ACAT2), and ATP binding cassette transporters 5 and 8 (ABCG5/8) in a down trend, whereas it increased the gene expression of hepatic cholesterol-7α -hydroxylase (CYP7A1). It was concluded that Tartary buckwheat protein was at least one of the active ingredients in Tartary buckwheat flour to lower plasma TC, mainly mediated by enhancing the excretion of bile acids via up-regulation of hepatic CYP7A1 and also by inhibiting the absorption of dietary

  3. Active-R filter

    DOEpatents

    Soderstrand, Michael A.

    1976-01-01

    An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

  4. International Conference: Paraoxonases - Basic and Clinical Directions of Current Research (1st) Held in Ann Arbor, Michigan on April 22-24, 2004

    DTIC Science & Technology

    2005-04-01

    HDL cholesterol levels but prevented impairment of LCAT activity when plasma was exposed to oxidative stress. Excess PON 1 inhibited...variation at the PON- 1 gene locus in terms of their effect on HDL - cholesterol levels and on the carotid intima media thickness (IMT), a surrogate...positive correlation between PON- 1 levels and activity and HDL - cholesterol (PON- 1 levels : r=0.37, pɘ.001; paraoxonase activity: r=0.23, p=0.01;

  5. Comparison of effects of vegetable protein diet and animal protein diet on the initiation of anemia during vigorous physical training (sports anemia) in dogs and rats.

    PubMed

    Yamada, T; Tohori, M; Ashida, T; Kajiwara, N; Yoshimura, H

    1987-04-01

    The effect of the quality of dietary protein on the initiation of temporary anemia during vigorous physical training (sports anemia) was studied in dogs and rats. In the dog experiment, one group of dogs was fed a crude animal protein (AP) diet and the other a crude vegetable protein (VP) diet. After 6 weeks on the diets in a sedentary state (rest period), all the dogs were forced to run every day for two weeks (exercise period). The rat experiment was carried out using purified nutrient mixtures. Casein (C) was used as AP and gluten (G) as VP. Feeding was done for two weeks in two series with two diet groups of rats. One series was 15% protein feeding (15% C and 15% G) groups and the other 24% protein feeding (24% C and 24% G) groups. In each group, one group remained in a sedentary state (rest group), and the other ran vigorously on a treadmill every day for one week (exercise group). In a sedentary state, there was a slight tendency for the hemoglobin content or erythrocyte count to be reduced, even when the values remained within the normal range, in dogs and rats fed VP. On the other hand, after vigorous running, significant anemia (reduction of hemoglobin) appeared in the VP diet dogs and in all exercise rat groups except the 24% C group. It was confirmed that the anemia was caused by a reduction of erythrocyte resistance to hemolysis, which was closely related to changes in the lipid composition of blood (serum and especially erythrocytes). The change in lipid profile revealed by the experiments was a reduction of free cholesterol in blood associated with an increase of lysolecithin in dogs during the exercise period and in the rat exercise groups. It was suggested that repeated physical exercise increased the activity of LCAT (lecithin-cholesterol-acyltransferase) in the liver, spleen, etc., resulting in the above changes in lipid patterns in the blood. In dogs of AP and rats of 24% C, however, those changes in lipid pattern caused by exercise and sports

  6. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids

    SciTech Connect

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. T.; Tonge, Peter J.; Seeliger, Jessica C.

    2015-09-08

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids know as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinase PknB modifies PapA5 on three Thr residues, including two (T196, T198) located on an unresolved loop. These results clarify the DIM biosynthetic pathway and suggest possible mechanisms by which DIM biosynthesis may be regulated by the post-translational modification of PapA5.

  7. Get Active

    MedlinePlus

    ... Basics: Health Benefits What are the benefits of physical activity? Physical activity increases your chances of living longer. ... pain Help you feel better about yourself Is physical activity for everyone? Yes! Physical activity is good for ...

  8. The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics.

    PubMed

    Koetsier, Martijn J; Gombert, Andreas K; Fekken, Susan; Bovenberg, Roel A L; van den Berg, Marco A; Kiel, Jan A K W; Jekel, Peter A; Janssen, Dick B; Pronk, Jack T; van der Klei, Ida J; Daran, Jean-Marc

    2010-01-01

    Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the beta-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

  9. Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

    PubMed

    Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

    2014-09-05

    Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.

  10. Perennial peanut (Arachis glabrata Benth.) leaves contain hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase activity and accumulate hydroxycinnamoyl-tartaric acid esters.

    PubMed

    Sullivan, Michael L

    2014-05-01

    Many plants accumulate hydroxycinnamoyl esters to protect against abiotic and biotic stresses. Caffeoyl esters in particular can be substrates for endogenous polyphenol oxidases (PPOs). Recently, we showed that perennial peanut (Arachis glabrata Benth.) leaves contain PPO and identified one PPO substrate, caftaric acid (trans-caffeoyl-tartaric acid). Additional compounds were believed to be cis- and trans-p-coumaroyl tartaric acid and cis- and trans-feruloyl-tartaric acid, but lack of standards prevented definitive identifications. Here we characterize enzymatic activities in peanut leaves to understand how caftaric acid and related hydroxycinnamoyl esters are made in this species. We show that peanut leaves contain a hydroxycinnamoyl-CoA:tartaric acid hydroxycinnamoyl transferase (HTT) activity capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from CoA to tartaric acid (specific activities of 11 ± 2.8, 8 ± 1.8, 4 ± 0.8 pkat mg(-1) crude protein, respectively). The HTT activity was used to make cis- and trans-p-coumaroyl- and -feruloyl-tartaric acid in vitro. These products allowed definitive identification of the corresponding cis- and trans-hydroxycinnamoyl esters extracted from leaves. We tentatively identified sinapoyl-tartaric acid as another major phenolic compound in peanut leaves that likely participates in secondary reactions with PPO-generated quinones. These results suggest hydroxycinnamoyl-tartaric acid esters are made by an acyltransferase, possibly a BAHD family member, in perennial peanut. Identification of a gene encoding HTT and further characterization of the enzyme will aid in identifying determinants of donor and acceptor substrate specificity for this important class of biosynthetic enzymes. An HTT gene could also provide a means by genetic engineering for producing caffeoyl- and other hydroxycinnamoyl-tartaric acid esters in forage crops that lack them.

  11. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

    PubMed Central

    MacPhee, C H; Moores, K E; Boyd, H F; Dhanak, D; Ife, R J; Leach, C A; Leake, D S; Milliner, K J; Patterson, R A; Suckling, K E; Tew, D G; Hickey, D M

    1999-01-01

    A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine. PMID:10024526

  12. Fructose supplementation impairs rat liver autophagy through mTORC activation without inducing endoplasmic reticulum stress.

    PubMed

    Baena, Miguel; Sangüesa, Gemma; Hutter, Natalia; Sánchez, Rosa M; Roglans, Núria; Laguna, Juan C; Alegret, Marta

    2015-02-01

    Supplementation with 10% liquid fructose to female rats for 2weeks caused hepatic steatosis through increased lipogenesis and reduced peroxisome proliferator activated receptor (PPAR) α activity and fatty acid catabolism, together with increased expression of the spliced form of X-binding protein-1 (Rebollo et al., 2014). In the present study, we show that some of these effects are preserved after sub-chronic (8weeks) fructose supplementation, specifically increased hepatic expression of lipid synthesis-related genes (stearoyl-CoA desaturase, ×6.7-fold; acetyl-CoA carboxylase, ×1.6-fold; glycerol-3-phosphate acyltransferase, ×1.65-fold), and reduced fatty acid β-oxidation (×0.77-fold), resulting in increased liver triglyceride content (×1.69-fold) and hepatic steatosis. However, hepatic expression of PPARα and its target genes was not modified and, further, livers of 8-week fructose-supplemented rats showed no sign of unfolded protein response activation, except for an increase in p-IRE1 levels. Hepatic mTOR phosphorylation was enhanced (×1.74-fold), causing an increase in the phosphorylation of UNC-51-like kinase 1 (ULK-1) (×2.8-fold), leading to a decrease in the ratio of LC3B-II/LC3B-I protein expression (×0.39-fold) and an increase in the amount of the autophagic substrate p62, indicative of decreased autophagy activity. A harmful cycle may be established in the liver of 8-week fructose-supplemented rats where lipid accumulation may cause defective autophagy, and reduced autophagy may result in decreased free fatty acid formation from triglyceride depots, thus reducing the substrates for β-oxidation and further increasing hepatic steatosis. In summary, the length of supplementation is a key factor in the metabolic disturbances induced by fructose: in short-term studies, PPARα inhibition and ER stress induction are critical events, whereas after sub-chronic supplementation, mTOR activation and autophagy inhibition are crucial.

  13. Activation detector

    DOEpatents

    Bell, Zane William [Oak Ridge, TN; Boatner, Lynn Allen [Oak Ridge, TN

    2009-12-08

    A method of detecting an activator, the method including impinging with an activator a receptor material lacking a photoluminescent material and generating a by-product of a radioactive decay due to the activator impinging the reeptor material. The method further including, generating light from the by-product via the Cherenkov effect and identifying a characteristic of the activator based on the light.

  14. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP.

    PubMed

    Wilcox, L J; Borradaile, N M; de Dreu, L E; Huff, M W

    2001-05-01

    The citrus flavonoids, naringenin and hesperetin, lower plasma cholesterol in vivo. However, the underlying mechanisms are not fully understood. The ability of these flavonoids to modulate apolipoprotein B (apoB) secretion and cellular cholesterol homeostasis was determined in the human hepatoma cell line, HepG2. apoB accumulation in the media decreased in a dose-dependent manner following 24-h incubations with naringenin (up to 82%, P < 0.00001) or hesperetin (up to 74%, P < 0.002). Decreased apoB secretion was associated with reduced cellular cholesteryl ester mass. Cholesterol esterification was decreased, dose-dependently, up to 84% (P < 0.0001) at flavonoid concentrations of 200 microM. Neither flavonoid demonstrated selective inhibition of either form of acyl CoA:cholesterol acyltransferase (ACAT) as determined using CHO cells stably transfected with either ACAT1 or ACAT2. However, in HepG2 cells, ACAT2 mRNA was selectively decreased (- 50%, P < 0.001) by both flavonoids, whereas ACAT1 mRNA was unaffected. In addition, naringenin and hesperetin decreased both the activity (- 20% to - 40%, P < 0.00004) and expression (- 30% to - 40%, P < 0.02) of microsomal triglyceride transfer protein (MTP). Both flavonoids caused a 5- to 7-fold increase (P < 0.02) in low density lipoprotein (LDL) receptor mRNA, which resulted in a 1.5- to 2-fold increase in uptake and degradation of (125)I-LDL. We conclude that both naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by 1) reduced activities of ACAT1 and ACAT2, 2) a selective decrease in ACAT2 expression, and 3) reduced MTP activity. Together with an enhanced expression of the LDL receptor, these mechanisms may explain the hypocholesterolemic properties of the citrus flavonoids.

  15. Cholesterol-lowering activity of sesamin is associated with down-regulation on genes of sterol transporters involved in cholesterol absorption.

    PubMed

    Liang, Yin Tong; Chen, Jingnan; Jiao, Rui; Peng, Cheng; Zuo, Yuanyuan; Lei, Lin; Liu, Yuwei; Wang, Xiaobo; Ma, Ka Ying; Huang, Yu; Chen, Zhen-Yu

    2015-03-25

    Sesame seed is rich in sesamin. The present study was to (i) investigate the plasma cholesterol-lowering activity of dietary sesamin and (ii) examine the interaction of dietary sesamin with the gene expression of sterol transporters, enzymes, receptors, and proteins involved in cholesterol metabolism. Thirty hamsters were divided into three groups fed the control diet (CON) or one of two experimental diets containing 0.2% (SL) and 0.5% (SH) sesamin, respectively, for 6 weeks. Plasma total cholesterol (TC) levels in hamsters given the CON, SL, and SH diets were 6.62 ± 0.40, 5.32 ± 0.40, and 5.00 ± 0.44 mmol/L, respectively, indicating dietary sesamin could reduce plasma TC in a dose-dependent manner. Similarly, the excretion of total fecal neutral sterols was dose-dependently increased with the amounts of sesamin in diets (CON, 2.65 ± 0.57; SL, 4.30 ± 0.65; and SH, 5.84 ± 1.27 μmol/day). Addition of sesamin into diets was associated with down-regulation of mRNA of intestinal Niemann-Pick C1 like 1 protein (NPC1L1), acyl-CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP-binding cassette transporters subfamily G members 5 and 8 (ABCG5 and ABCG8). Results also showed that dietary sesamin could up-regulate hepatic cholesterol-7α-hydroxylase (CYP7A1), whereas it down-regulated hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and liver X receptor alpha (LXRα). It was concluded that the cholesterol-lowering activity of sesamin was mediated by promoting the fecal excretion of sterols and modulating the genes involved in cholesterol absorption and metabolism.

  16. Catalyst activator

    DOEpatents

    McAdon, Mark H.; Nickias, Peter N.; Marks, Tobin J.; Schwartz, David J.

    2001-01-01

    A catalyst activator particularly adapted for use in the activation of metal complexes of metals of Group 3-10 for polymerization of ethylenically unsaturated polymerizable monomers, especially olefins, comprising two Group 13 metal or metalloid atoms and a ligand structure including at least one bridging group connecting ligands on the two Group 13 metal or metalloid atoms.

  17. Outdoor Activities.

    ERIC Educational Resources Information Center

    Minneapolis Independent School District 275, Minn.

    Twenty-four activities suitable for outdoor use by elementary school children are outlined. Activities designed to make children aware of their environment include soil painting, burr collecting, insect and pond water collecting, studies of insect galls and field mice, succession studies, and a model of natural selection using dyed toothpicks. A…

  18. Physical activity

    MedlinePlus

    ... activity -- which includes an active lifestyle and routine exercise -- plus eating well, is the best way to stay healthy. ... goal. Your goal might be to: Manage a health condition Reduce stress ... other benefits, such as: Better control of your weight and ...

  19. Astronomy Activities.

    ERIC Educational Resources Information Center

    Greenstone, Sid

    This document consists of activities and references for teaching astronomy. The activities (which include objectives, list of materials needed, and procedures) focus on: observing the Big Dipper and locating the North Star; examining the Big Dipper's stars; making and using an astrolabe; examining retograde motion of Mars; measuring the Sun's…

  20. (−)-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Shih, Chun-Ching; Wu, Jin-Bin; Jian, Jia-Ying; Lin, Cheng-Hsiu; Ho, Hui-Ya

    2015-01-01

    The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−)-epicatechin-3-O-β-d-allopyranoside (BB) from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD) or low-fat diet (control diet, CD). After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi) or fenofibrate (Feno) or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG), and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4) were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate) protein kinase (phospho-AMPK). BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK), which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα), and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT) mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model. PMID:26492243

  1. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl Beta-Diol Lipids

    PubMed Central

    Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John E.; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. Todd; Tonge, Peter J.; Seeliger, Jessica C.

    2015-01-01

    Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in regulation DIM biosynthesis. PMID:26271001

  2. Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids.

    PubMed

    Touchette, Megan H; Bommineni, Gopal R; Delle Bovi, Richard J; Gadbery, John E; Nicora, Carrie D; Shukla, Anil K; Kyle, Jennifer E; Metz, Thomas O; Martin, Dwight W; Sampson, Nicole S; Miller, W Todd; Tonge, Peter J; Seeliger, Jessica C

    2015-09-08

    Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.

  3. Activated Charcoal

    MedlinePlus

    ... is used to treat poisonings, reduce intestinal gas (flatulence), lower cholesterol levels, prevent hangover, and treat bile ... lower cholesterol levels in the blood. Decreasing gas (flatulence). Some studies show that activated charcoal is effective ...

  4. Activity Theory.

    ERIC Educational Resources Information Center

    Koschmann, Timothy; Roschelle, Jeremy; Nardi, Bonnie A.

    1998-01-01

    Includes three articles that discuss activity theory, based on "Context and Consciousness." Topics include human-computer interaction; computer interfaces; hierarchical structuring; mediation; contradictions and development; failure analysis; and designing educational technology. (LRW)

  5. Plant metacaspase activation and activity.

    PubMed

    Minina, Elena A; Stael, Simon; Van Breusegem, Frank; Bozhkov, Peter V

    2014-01-01

    Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of metacaspases and their molecular function in plant biology requires a set of robust methods for detection of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe methods for purification of recombinant metacaspases, measurement of enzymatic activity of recombinant and endogenous metacaspases in vitro and in cell lysates, respectively, and finally detection of metacaspase activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-free production of substrate protein followed by proteolysis with recombinant metacaspase is presented. These methods have been originally developed for type II metacaspases from Arabidopsis and Norway spruce (Picea abies), but they can be used as templates for type I metacaspases, as well as for type II metacaspases from other species.

  6. Active colloids

    NASA Astrophysics Data System (ADS)

    Aranson, Igor S.

    2013-01-01

    A colloidal suspension is a heterogeneous fluid containing solid microscopic particles. Colloids play an important role in our everyday life, from food and pharmaceutical industries to medicine and nanotechnology. It is useful to distinguish two major classes of colloidal suspensions: equilibrium and active, i.e., maintained out of thermodynamic equilibrium by external electric or magnetic fields, light, chemical reactions, or hydrodynamic shear flow. While the properties of equilibrium colloidal suspensions are fairly well understood, active colloids pose a formidable challenge, and the research is in its early exploratory stage. One of the most remarkable properties of active colloids is the possibility of dynamic self-assembly, a natural tendency of simple building blocks to organize into complex functional architectures. Examples range from tunable, self-healing colloidal crystals and membranes to self-assembled microswimmers and robots. Active colloidal suspensions may exhibit material properties not present in their equilibrium counterparts, e.g., reduced viscosity and enhanced self-diffusivity, etc. This study surveys the most recent developments in the physics of active colloids, both in synthetic and living systems, with the aim of elucidation of the fundamental physical mechanisms governing self-assembly and collective behavior.

  7. Active microwaves

    NASA Technical Reports Server (NTRS)

    Evans, D.; Vidal-Madjar, D.

    1994-01-01

    Research on the use of active microwaves in remote sensing, presented during plenary and poster sessions, is summarized. The main highlights are: calibration techniques are well understood; innovative modeling approaches have been developed which increase active microwave applications (segmentation prior to model inversion, use of ERS-1 scatterometer, simulations); polarization angle and frequency diversity improves characterization of ice sheets, vegetation, and determination of soil moisture (X band sensor study); SAR (Synthetic Aperture Radar) interferometry potential is emerging; use of multiple sensors/extended spectral signatures is important (increase emphasis).

  8. Activated Sludge.

    ERIC Educational Resources Information Center

    Saunders, F. Michael

    1978-01-01

    Presents the 1978 literature review of wastewater treatment. This review covers: (1) activated sludge process; (2) process control; (3) oxygen uptake and transfer; (4) phosphorus removal; (5) nitrification; (6) industrial wastewater; and (7) aerobic digestion. A list of 136 references is also presented. (HM)

  9. Activities: Spirolaterals.

    ERIC Educational Resources Information Center

    Brannan, Richard; McFadden, Scott

    1981-01-01

    A set of activities designed to help students discover properties about order-3 spirolaterals on square grid paper is presented. The materials are prepared on worksheets designed for easy duplication. The lessons can lead to investigations involving spirolaterals of many other orders and shapes. (MP)

  10. Leaf Activities.

    ERIC Educational Resources Information Center

    Mingie, Walter

    Leaf activities can provide a means of using basic concepts of outdoor education to learn in elementary level subject areas. Equipment needed includes leaves, a clipboard with paper, and a pencil. A bag of leaves may be brought into the classroom if weather conditions or time do not permit going outdoors. Each student should pick a leaf, examine…

  11. Learning Activities.

    ERIC Educational Resources Information Center

    Tipton, Tom, Ed.

    1983-01-01

    Presents a flow chart for naming inorganic compounds. Although it is not necessary for students to memorize rules, preliminary skills needed before using the chart are outlined. Also presents an activity in which the mass of an imaginary atom is determined using lead shot, Petri dishes, and a platform balance. (JN)

  12. Activity report

    SciTech Connect

    Yu, S W

    2008-08-11

    This report is aimed to show the author's activities to support the LDRD. The title is 'Investigation of the Double-C Behavior in the Pu-Ga Time-Temperature-Transformation Diagram' The sections are: (1) Sample Holder Test; (2) Calculation of x-ray diffraction patterns; (3) Literature search and preparing publications; (4) Tasks Required for APS Experiments; and (5) Communications.

  13. Antimicrobial Activity.

    PubMed

    2016-01-01

    Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with conventional antimicrobials. A method using scanning electron microscope (SEM) to study the morphology of the bacterial and fungal microbes and thus determining antimicrobial activity is presented in the chapter.

  14. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  15. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated