Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.     

Surfactant phospholipid metabolism

Pulmonary surfactant is essential for life and is composed of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration. The molecular composition of surfactant depends on highly integrated and regulated processes involving its biosynthesis, remodeling, degradation, and intracellular trafficking. Despite its multicomponent composition, the study of surfactant phospholipid metabolism has focused on two predominant components, disaturated phosphatidylcholine that confers surface-tension lowering activities, and phosphatidylglycerol, recently implicated in innate immune defense. Future studies providing a better understanding of the molecular control and physiological relevance of minor surfactant lipid components are needed. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Concurrent modulation of extracellular levels of noradrenaline and cAMP during stress and by anxiogenic- or anxiolytic-like neuropeptides in the prefrontal cortex of awake rats

The purpose of this study was to examine the effects of stress and the role of locally infused anxiogenic-like neuropeptides galanin, CCK-8, vasopressin, substance P and neurokinin A, and anxiolytic-like peptides NPY, nociceptin/orphanin FQ, somatostatin and neurotensin, on modulation of noradrenaline (NA) and cAMP efflux monitored simultaneously by microdialysis in the medial prefronatal cortex of awake rats. Concentrations of cAMP were determined by a newly developed method based on derivatization of cAMP with 2-chloroacetaldehyde followed by HPLC with fluorescence detection. Local infusion of forskolin (10 and 30 μM) dose-dependently increased the cAMP levels to 417% and 1050% of the control group, respectively. Similarly, local infusion of NA (10 μM) increased the cAMP to the peak level of 168%. A 5-min tail pinch and a 10-min swim stress rapidly increased the NA and cAMP levels to 167% and 203% (NA) and 141% and 161% (cAMP), respectively. Infusion of galanin and CCK-8 (0.5 nmol, and 1.5 nmol/0.5 μl) dose-dependently increased NA to the peak levels of 191% and 179% and cAMP levels to 174% and 166%, respectively. The peak levels following infusions of vasopressin, substance P and neurokinin A were 91%, 135% and 86% for NA and 131%, 83% and 76% for cAMP, respectively. Infusions of anxiolytic-like peptides at highest concentrations significantly increased (NPY, 136%) or decreased (nociceptin, 71%; somatostatin, 86%) the NA levels, whereas neurotensin had no effect. The cAMP levels decreased to 86% (NPY, neurotensin), 78% (nociceptin), somatostatin infusion was without effect. The present findings confirmed a close correlation between the stress-induced increases in prefrontal cortical NA and cAMP levels, as well as, concurrent changes in NA and cAMP levels following infusions of galanin and CCK-8 (increased levels) and nociceptin/orphanin FQ (decreased levels). Infusions of other neuropeptides showed a more complex pattern of NA and cAMP responses.     

Phosphatidate phosphatase,a key regulator of lipid homeostasis

Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The cellular amounts of these lipid intermediates influence the synthesis of triacylglycerol and the pathways by which membrane phospholipids are synthesized. Physiological functions affected by PAP activity include phospholipid synthesis gene expression, nuclear/endoplasmic reticulum membrane growth, lipid droplet formation, and vacuole homeostasis and fusion. Yeast lacking Pah1p PAP activity are acutely sensitive to fatty acid-induced toxicity and exhibit respiratory deficiency. PAP is distinguished in its cellular location, catalytic mechanism, and physiological functions from Dpp1p and Lpp1p lipid phosphate phosphatases that utilize a variety of substrates that include phosphatidate. Phosphorylation/dephosphorylation is a major mechanism by which Pah1p PAP activity is regulated. Pah1p is phosphorylated by cytosolic-associated Pho85p–Pho80p, Cdc28p-cyclin B, and protein kinase A and is dephosphorylated by the endoplasmic reticulum-associated Nem1p–Spo7p phosphatase. The dephosphorylation of Pah1p stimulates PAP activity and facilitates the association with the membrane/phosphatidate allowing for its reaction and triacylglycerol synthesis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line

Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A₂ and C). A phospholipase C–diacylglycerol lipase–monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.     

Interaction of the C-terminal domain of Bcl-2 family proteins with model membranes

Bcl-2 family proteins are involved in the cell homeostasis by regulating programmed cell death. Some of these proteins promote apoptosis, while others inhibit the same process. The C-terminal hydrophobic domain of some of these proteins is predicted to be involved in anchoring them to a variety of cell membranes, such as mitochondrial, endoplasmic reticulum and nuclear membranes. We have used five synthetic peptides imitating the C-terminal domain from both anti-apoptotic (Bcl-2) and pro-apoptotic members (Bak, Bax, and two mutants of this last protein) of this family to study their interaction with model membranes. Some differences were detected in the interaction with these peptides. The addition of all the peptides to large unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein to different degrees, so that fluidity and the increase in negative curvature favoured the extent in the release of carboxyfluorescein. Bcl-2-C and Bax-C peptides produced the highest release levels in most cases, while BaxS184K-C was the least efficient in this respect. These results indicate that these C-terminal domains are able to insert themselves in the membranes, each in a different way that is probably related with their different way which can be related to their differing locations within the cell and their different roles in regulating apoptosis.     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

Lipid Signaling in Experimental Epilepsy

Glutamate release activates signaling pathways important for learning and memory, and over-stimulation of these pathways during seizures leads to aberrant synaptic plasticity associated with hyper-excitable, seizure-prone states. Seizures induce rapid accumulation of membrane lipid-derived fatty acids at the synapses which, evidence suggests, regulate maladaptive connectivity. Here we give an overview of the significance of the arachidonyl- and inositol-derived messengers, prostaglandins (PGs) and diacylglycerol (DAG), in experimental models of epilepsy. We use studies conducted in our own laboratory to highlight the pro-epileptogenic role of cyclooxygenase-2 (COX-2) and its products, the PGs, and we discuss the possible mechanisms by which PGs may regulate membrane excitability and synaptic transmission at the cellular level. We conclude with a discussion of AA-DAG signaling in synaptic plasticity and seizure susceptibility with an emphasis on recent studies in our laboratory involving DAG kinase ε (DGKε)-knockout mice.     

The bovine vitreous-derived lipid factor (bVLF) is a powerful inhibitor of retinal pigmented epithelial (hRPE) cell proliferation

Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca(2+)-mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca(2+)-mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose-dependent manner, a Ca(2+) mobilization from PA-sensitive intracellular stores [non-Ins(1,4,5)P(3)-sensitive stores], in which extracellular Ca(2+) participated. The increase in intracellular Ca(2+) was associated with a dose-dependent inhibiting effect on cell proliferation. At a dose of 10 microg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3- or the 6-day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR-T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF-, bFGF-, IGF-I-, PDGF-, HGF- and VEGF-induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS-induced hRPE cell proliferation. bVLF, at a concentration of 10 microg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6-day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE-associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.     

Signaling Cascade of Diacylglycerol Kinase �� in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase C��4/Diacylglycerol Kinase ��/Protein Kinase C��

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe. (J Histochem Cytochem 58:119–129, 2010)