Regulation Of Phospholipid Biosynthesis Research Articles

A mouse model of gestational diabetes shows dysregulated lipid metabolism post-weaning, after return to euglycaemia

BackgroundGestational diabetes is associated with increased risk of type 2 diabetes mellitus and cardiovascular disease for the mother in the decade after delivery. However, the molecular mechanisms that drive these effects are unknown. Recent studies in humans have shown that lipid metabolism is dysregulated before diagnosis of and during gestational diabetes and we have shown previously that lipid metabolism is also altered in obese female mice before, during and after pregnancy. These observations led us to the hypothesis that this persistent dysregulation reflects an altered control of lipid distribution throughout the organism.MethodsWe tested this in post-weaning (PW) dams using our established mouse model of obese GDM (high fat, high sugar, obesogenic diet) and an updated purpose-built computational tool for plotting the distribution of lipid variables throughout the maternal system (Lipid Traffic Analysis v2.3).ResultsThis network analysis showed that unlike hyperglycaemia, lipid distribution and traffic do not return to normal after pregnancy in obese mouse dams. A greater range of phosphatidylcholines was found throughout the lean compared to obese post-weaning dams. A range of triglycerides that were found in the hearts of lean post-weaning dams were only found in the livers of obese post-weaning dams and the abundance of odd-chain FA-containing lipids differed locally in the two groups. We have therefore shown that the control of lipid distribution changed for several metabolic pathways, with evidence for changes to the regulation of phospholipid biosynthesis and FA distribution, in a number of tissues.ConclusionsWe conclude that the control of lipid metabolism is altered following an obese pregnancy. These results support the hypothesis that obese dams that developed GDM maintain dysregulated lipid metabolism after pregnancy even when glycaemia returned to normal, and that these alterations could contribute to the increased risk of later type 2 diabetes and cardiovascular disease.

The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K

Background : Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods : We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results : CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions : Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.

The herpes simplex virus 1 Us3 kinase is involved in assembly of membranes needed for viral envelopment and in distribution of glycoprotein K.

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in cell nuclei, released into the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope. Alternatively, capsids gain access to the cytoplasm via dilated nuclear pores. They are enveloped by Golgi membranes. Us3 is a non-essential viral kinase that is involved in nucleus-to-cytoplasm translocation, preventing apoptosis and regulation of phospholipid-biosynthesis. Us3-deletion mutants (HSV-1∆Us3) accumulate in the perinuclear space. Nuclear and Golgi membranes proliferate, and homogeneous, proteinaceous structures of unknown identity are deposited in nuclei and cytoplasm. Glycoprotein K (gK), a highly hydrophobic viral protein, is essential for production of infectious progeny virus but, according to the literature, exclusively vital for envelopment of capsids by Golgi membranes. In the absence of Us3, virions remain stuck in the perinuclear space but mature to infectivity without reaching Golgi membranes, suggesting further function of gK than assumed. Methods: We constructed a HSV-1∆Us3 mutant designated CK177∆Us3gK-HA, in which gK was hemagglutinin (HA) epitope-tagged in order to localize gK by immunolabeling using antibodies against HA for light and electron microscopy. Results: CK177∆Us3gK-HA-infected Vero cells showed similar alterations as those reported for other HSV-1∆Us3, including accumulation of virions in the perinuclear space, overproduction of nuclear and Golgi membranes containing electron dense material with staining property of proteins. Immunolabeling using antibodies against HA revealed that gK is overproduced and localized at nuclear membranes, perinuclear virions stuck in the perinuclear space, Golgi membranes and on protein deposits in cytoplasm and nuclei. Conclusions: Us3 is involved in proper assembly of membranes needed for envelopment and incorporation of gK. Without Us3, virions derived by budding at nuclear membranes remain stuck in the perinuclear space but incorporate gK into their envelope to gain infectivity.

Saccharomyces cerevisiaeEssential Genes with an Opi− Phenotype

The overproduction and secretion of inositol (i.e., Opi−) phenotype is associated with defects in regulation of phospholipid biosynthesis in yeast. Here we report a screen of the essential yeast gene set using a conditional-expression library. This screen identified novel functions previously unknown to affect phospholipid synthesis.

Regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C through the transcriptional repression of upstream activating sequence inositol containing genes

The regulation of phospholipid biosynthesis in Saccharomyces cerevisiae through cis-acting upstream activating sequence inositol (UASino) and trans-acting elements, such as the INO2–INO4 complex and OPI1 by inositol supplementation in growth is thoroughly studied. In this study, we provide evidence for the regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C (PLC) through UASino and the trans-acting elements. Gene expression analysis and radiolabelling experiments demonstrated that the overexpression of rice PLC in yeast cells altered phospholipid biosynthesis at the levels of transcriptional and enzyme activity. This is the first report implicating PLC in the direct regulation of lipid biosynthesis.

Inhibition of phospholipid biosynthesis decreases the activity of the tombusvirus replicase and alters the subcellular localization of replication proteins

Replication of plus-strand RNA viruses depends on lipids present in cellular membranes. Recent genome-wide screens have revealed that eight phospholipid biosynthesis genes affected the replication of Tomato bushy stunt virus (TBSV) in yeast model host. To test the importance of phospholipids in TBSV replication, we studied one of the identified genes, namely INO2, which forms a heterodimer with Ino4, and is a transcription activator involved in regulation of phospholipid biosynthesis. Deletion of INO2, or double deletion of INO2/INO4, reduced TBSV replication and inhibited the activity of the viral replicase complex. In addition, the stability of the viral replication protein is decreased as well as the localization pattern of the viral protein changed dramatically in ino2∆ino4∆ yeast. Over-expression of Opi1, a repressor of Ino2 and phospholipid biosynthesis, also inhibited TBSV RNA accumulation. In contrast, over-expression of Ino2 stimulated TBSV RNA accumulation. We also observed an inhibitory effect on Flock house virus (FHV) replication and the reduced stability of the FHV replication protein in ino2∆ino4∆ yeast. These data are consistent with the important role of phospholipids in RNA virus replication.

Regulation of Phospholipid Biosynthesis in Arabidopsis

Phospholipids are major constituents of the membranes that separate cells from their environment and compartmentalize eukaryotic cells into functional units. In yeast ( Saccharomyces cerevisiae ), the expression of genes involved in phospholipid biosynthesis is regulated by a mechanism that responds

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

The yeast VAP homolog Scs2p has a phosphoinositide-binding ability that is correlated with its activity

The yeast VAMP-associated protein (VAP) homolog Scs2p is an endoplasmic reticulum (ER)/nuclear membrane protein that binds to an FFAT (diphenylalanine in an acidic tract) motif found in various lipid-metabolic proteins, including Opi1p, a negative regulator of phospholipid biosynthesis. Here, we show that Scs2p is a novel phosphoinositide-binding protein that can bind to phosphatidylinositol monophosphates and bisphosphates in vitro. The phosphoinositide-binding domain was assigned to the N-terminal major sperm protein (MSP) domain which also contains the FFAT-binding domain. When several lysine residues in the MSP domain were substituted for alanine, the resulting mutant Scs2 proteins lost the phosphoinositide-binding ability and failed to complement the inositol auxotrophy of an scs2 deletion strain. However, the mutant proteins still localized in the ER/nuclear membrane, in a similar manner to wild-type Scs2p. These results suggest the possibility that Scs2p activity is regulated by phosphoinositides to coordinate phospholipid biosynthesis in response to changes in phospholipid composition.

Regulation of phospholipid biosynthesis by phosphatidylinositol transfer protein Sec14p and its homologues

Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism. The INO1 gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in the sec14Delta cki1Delta strain (SEC14 encodes the major yeast phosphatidylinositol transfer protein; CKI1 encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis [Henry, S.A. & Patton-Vogt, J.L. (1998) Prog. Nucleic Acid Res. Mol. Biol.61, 133-179].

Cellular Regulation of Phospholipid Biosynthesis

How do cells homeostatically regulate the lipid composition of their membranes? Phospholipid metabolism in yeast is coordinately repressed by the transcription factor Opi1p, which in turn is activated by extracellular inositol. Loewen et al. now show that there is a pool of phosphatidic acid (PA) on the endoplasmic reticulum (ER) that is highly sensitive to growth conditions, in particular to the presence of exogenous inositol. This PA is directly sensed by the soluble transcriptional repressor Opi1p, which binds to PA in the ER and prevents it from entering the nucleus and altering transcription. When inositol is restored to cells, the Opi1p-associated PA is metabolized, which releases the Opi1p from the ER and allows its entry into the nucleus. C. J. Loewen, M. L. Gaspar, S. A. Jesch, C. Delon, N. T. Ktistakis, S. A. Henry, T. P. Levine, Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid. Science 304, 1644-1647 (2004). [Abstract] [Full Text]

Cellular Regulation of Phospholipid Biosynthesis

Cellular Regulation of Phospholipid Biosynthesis

Cellular Regulation of Phospholipid Biosynthesis

Cellular Regulation of Phospholipid Biosynthesis

Possible involvement of Ca2+/calmodulin-dependent protein kinase in the regulation of phospholipid biosynthesis in Microsporum gypseum.

The mechanism of action of Ca2+/calmodulin on phospholipid synthesis in Microsporum gypseum has been studied. These second messengers were observed to mediate their function through phosphorylation mechanism as altered protein kinase activity was seen in calcium/trifluoperazine (calmodulin antagonist) grown cells. The activity of protein kinase was dependent on calcium (200 microm) and calmodulin (1 microm). In vitro studies of phosphorylation and dephosphorylation in relation to phospholipid synthesis in Microsporum gypseum have been carried out. Addition of KN-62 (a specific inhibitor of Ca2+/calmodulin-dependent protein kinases) and polyclonal antibodies raised against purified Ca2+/calmodulin-kinase (CaMPK) of M. gypseum in the cell extract, leads to the inhibition in the incorporation of labelled acetate into total phospholipids in this fungus. These results suggest a possible involvement of Ca2+/calmodulin via Ca2+/calmodulin-dependent phosphorylation in phospholipid synthesis in M. gypseum.

Regulation of phospholipid biosynthesis by Ca 2+ -calmodulin-dependent protein kinase inhibitors

Inhibitors of Ca 2+-calmodulin (CaM)-dependent protein kinases strongly modify phospholipid metabolism. Two compounds, KN62 and KT5926 recognized as blockers of Ca 2+ -CaM-dependent protein kinase II, induced a specific increase in phosphatidylserine (PtdSer) synthesis without noticeable changes in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) biosynthesis. The increase of PtdSer synthesis was dependent on the presence of Ca 2+ in the incubation medium and was impaired in cells whose Ca 2+ stores were depleted by pretreatment with CD3 mAb, thapsigargin or EGTA. The mechanism of the stimulation of PtdSer synthesis by these two compounds seems to involve an accumulation of Ca 2+ into the endoplasmic reticulum, possibly due to an increased activity of the endoplasmic reticulum Ca 2+ -ATPase. By contrast, ML-7 and ML-9, two inhibitors of the myosin light chain kinase (MLCK), another Ca 2+-CaM-dependent kinase, were both capable of increasing PtdSer synthesis and decreasing PtdCho and PtdEtn synthesis, reproducing the effect previously described with CaM-antagonists. The increase of PtdSer caused by ML-7 and ML-9 was Ca 2+-dependent while the inhibition of PtdCho and PtdEtn synthesis was not. The use of these four protein kinase inhibitors thus suggests the possible existence of two CaM-dependent pathways that differentially regulates phospholipid metabolism in T cells.

Regulation of Phospholipid Biosynthesis in the Yeast Saccharomyces cerevisiae

Phospholipids are key molecules that contribute to the structural definition of cells and that participate in the regulation of cellular processes. Phospholipid metabolism is a major activity that cells engage in throughout their growth. The yeast, Saccharomyces cerevisiae, serves as a model system in which to study the regulation of phospholipid synthesis and its regulation in eucaryotes. Its membranous organelles, the lipids that comprise these membranes, and the phospholipid biosynthetic pathways that generate these membranes typify eucaryotic cells (1, 2). Many of the structural genes encoding for the phospholipid biosynthetic enzymes have been cloned and characterized (Table I) (3–25), and a number of mutations in these genes have been isolated (3, 7–9, 11–13, 17, 23, 26–33). In addition, a number of the phospholipid biosynthetic enzymes have been purified and studied (Table I) (34–44). The characterization of the wild-type and mutant genes, as well as the gene products encoded by these alleles, has significantly advanced our understanding both of phospholipid biosynthesis and of its regulation. Results from these genetic, molecular, and biochemical studies have shown that the regulation of phospholipid synthesis is a complex, highly coordinated process. The mechanisms that govern this regulation mediate the mRNA and protein levels of the biosynthetic enzymes as well as their activity and localization (1, 2, 45). This review summarizes our current understanding of the regulation of phospholipid metabolism in S. cerevisiae with a particular focus on the regulation of the activity of the biosynthetic enzymes. For more comprehensive reviews, the reader is directed to recent articles by Paltauf et al. (2) and Greenberg and Lopes (45).

Genetic regulation of phospholipid biosynthesis in Saccharomyces cerevisiae.

INTRODUCTION 1 GENETIC REGULATION 2 Biosynthetic Pathways 2 Phosphatidic acid and CDP-diacylglycerol synthesis 2 Phosphatidylinositol branch 4 Phosphatidylcholine branch: de novo pathway 5 Phosphatidylcholine branch: salvage pathway 6 Inositol and choline transporters 6 Cardiolipin branch 7 Sphingolipids 8 Fatty acids 8 Cross-Pathway Regulation 8 Control by Factors Affecting Mitochondrial Development 9 LEVELS OF REGULATION 9 INOSITOL/CHOLINE RESPONSE 10 UASINO Regulatory Sequence 10 INO2 and INO4 Positive Regulatory Genes 11 OPI1 Negative Regulatory Gene 12 Other Regulatory Genes 13 Autoregulation of INO2 Expression 13 URS1 Regulatory Sequence 14 SIN3 and UME6 Global Negative Regulators 14 INO1 TRANSCRIPTION AND THE GENERAL TRANSCRIPTION MACHINERY 14 REGULATION OF PHOSPHOLIPID BIOSYNTHESIS IN OTHER FUNGI 15 Schizosaccharomyces pombe 15 Candida albicans 15 Neurospora crassa 16 CONCLUSIONS 16 ACKNOWLEDGMENTS 16 REFERENCES 16

Regulation of Phospholipid Biosynthesis in Saccharomyces cerevisiae by CTP

In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways. We examined the regulation of phosphatidylcholine synthesis by CTP. The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP. The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway. CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway. The apparent Km of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline. Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase. This was not due to a transcriptional repression mechanism. Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP. These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized. This regulation includes the supple of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway.

35] Phosphatidylserine synthase from yeast

This chapter describes the purification, reconstitution, and properties of the phosphatidylserine synthase. Phosphatidylserine synthase catalyzes the incorporation of serine into phosphatidylserine. The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae . Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae . Phosphatidylserine synthase expression is regulated by inositol and in concert with serine, ethanolamine, and choline. Microsomal-associated phosphatidylserine synthase has been purified to near homogeneity using CDPdiacylglycerol-sepharose affinity chromatography. CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi with the modifications of Carman and Fischl. The procedure for the electroblotting of phosphatidylserine synthase is similar to that used for the electroblotting of phosphatidylinositol synthase. The presence of CDPdiacylglycerol and cofactors in the renaturation buffer stabilizes phosphatidylserine synthase activity. The nitrocellulose paper is used for enzyme assays. The recovery of phosphatidylserine synthase activity is about 10%. This procedure allows the confirmation of the molecular weight of phosphatidylserine synthase. Pure phosphatidylserine synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol. Pure phosphatidylserine synthase can be used to synthesize radiolabeled phosphatidylserine from CDPdiacylglycerol and labeled serine. The enzyme can be used to synthesize various fatty acyl derivatives of phosphatidylserine using the appropriate fatty acyl derivatives of CDPdiacylglycerol as substrate.

Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity

Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.