Structural and Functional Characterization of Phosphatidylinositol-Phosphate Biosynthesis in Mycobacteria.

In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate-diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate-alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

Phosphatidylinositol synthesis at the endoplasmic reticulum.

Phosphatidylinositol (PI) is a minor phospholipid with a characteristic fatty acid profile; it is highly enriched in stearic acid at the sn-1 position and arachidonic acid at the sn-2 position. PI is phosphorylated into seven specific derivatives, and individual species are involved in a vast array of cellular functions including signalling, membrane traffic, ion channel regulation and actin dynamics. De novo PI synthesis takes place at the endoplasmic reticulum where phosphatidic acid (PA) is converted to PI in two enzymatic steps. PA is also produced at the plasma membrane during phospholipase C signalling, where hydrolysis of phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) leads to the production of diacylglycerol which is rapidly phosphorylated to PA. This PA is transferred to the ER to be also recycled back to PI. For the synthesis of PI, CDP-diacylglycerol synthase (CDS) converts PA to the intermediate, CDP-DG, which is then used by PI synthase to make PI. The de novo synthesised PI undergoes remodelling to acquire its characteristic fatty acid profile, which is altered in p53-mutated cancer cells. In mammals, there are two CDS enzymes at the ER, CDS1 and CDS2. In this review, we summarise the de novo synthesis of PI at the ER and the enzymes involved in its subsequent remodelling to acquire its characteristic acyl chains. We discuss how CDS, the rate limiting enzymes in PI synthesis are regulated by different mechanisms. During phospholipase C signalling, the CDS1 enzyme is specifically upregulated by cFos via protein kinase C.

Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms.

Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. Here, we report that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIα partially rescues the mitochondrial defects of pss RNAi. Together, our results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms.

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.

Structural basis for phosphatidylinositol-phosphate biosynthesis.

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol.

Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.

Phosphoinositide signalling in Drosophila.

Phosphoinositides (PtdInsPs) are lipids that mediate a range of conserved cellular processes in eukaryotes. These include the transduction of ligand binding to cell surface receptors, vesicular transport and cytoskeletal function. The nature and functions of PtdInsPs were initially elucidated through biochemical experiments in mammalian cells. However, over the years, genetic and cell biological analysis in a range of model organisms including S. cerevisiae, D. melanogaster and C. elegans have contributed to an understanding of the involvement of PtdInsPs in these cellular events. The fruit fly Drosophila is an excellent genetic model for the analysis of cell and developmental biology as well as physiological processes, particularly analysis of the complex relationship between the cell types of a metazoan in mediating animal physiology. PtdInsP signalling pathways are underpinned by enzymes that synthesise and degrade these molecules and also by proteins that bind to these lipids in cells. In this review we provide an overview of the current understanding of PtdInsP signalling in Drosophila. We provide a comparative genomic analysis of the PtdInsP signalling toolkit between Drosophila and mammalian systems. We also review some areas of cell and developmental biology where analysis in Drosophila might provide insights into the role of this lipid-signalling pathway in metazoan biology. This article is part of a Special Issue entitled Phosphoinositides.

Phosphoinositides: Lipids with informative heads and mastermind functions in cell division.

Phosphoinositides are low abundant but essential phospholipids in eukaryotic cells and refer to phosphatidylinositol and its seven polyphospho-derivatives. In this review, we summarize our current knowledge on phosphoinositides in multiple aspects of cell division in animal cells, including mitotic cell rounding, longitudinal cell elongation, cytokinesis furrow ingression, intercellular bridge abscission and post-cytokinesis events. PtdIns(4,5)P2production plays critical roles in spindle orientation, mitotic cell shape and bridge stability after furrow ingression by recruiting force generator complexes and numerous cytoskeleton binding proteins. Later, PtdIns(4,5)P2hydrolysis and PtdIns3P production are essential for normal cytokinesis abscission. Finally, emerging functions of PtdIns3P and likely PtdIns(4,5)P2have recently been reported for midbody remnant clearance after abscission. We describe how the multiple functions of phosphoinositides in cell division reflect their distinct roles in local recruitment of protein complexes, membrane traffic and cytoskeleton remodeling. This article is part of a Special Issue entitled Phosphoinositides.

Expression profiles and polymorphism analysis of CDIPT gene on Qinchuan cattle

Background CDIPT (CDP-diacylglycerol-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was found on the cytoplasmic side of the Golgi apparatus and the endoplasmic reticulum. It was an integral membrane protein performing the last step in the de novo biosynthesis of phosphatidylinositol (PtdIns). In recent years, PtdIns has been considered to play an essential role in energy metabolism, fatty acid metabolic pathway and intracellular signal transduction in eukaryotic cells. Results In this study, the results of real-time polymerase chain reaction (PCR) showed that the expression of CDIPT gene was remarkably different in diverse tissues. We also detected the polymorphism of bovine CDIPT gene and analyzed its association with body measurement and meat quality traits of Qinchuan cattle. Blood samples were obtained from 638 Qinchuan cattle aged from 18 to 24 months. DNA sequencing and PCR-restriction fragment length polymorphism (RFLP) were used to find CDIPT gene single nucleotide polymorphism (SNP). Three SNPs g.244T>C (NCBI: rs42069760), g.1496G>A and g.1514G>A were found in this study. g.244T>C located at 5'untranslated region (5'UTR) of exon 1 showed three genotypes: TT, TC and CC. g.1496G>A and g.1514G>A detected the first time were located in intron 3 and showed the same genotypes: GG, GA and AA. Conclusions Analysis results showed that these three SNPs were significantly associated with body measurement traits (BMTs) and meat quality traits (MQTs). We suggested that CDIPT gene may have potential effects on BMTs and MQTs and can be used for marker-assisted selection.

Polarized deposition of basement membrane proteins depends on Phosphatidylinositol synthase and the levels of Phosphatidylinositol 4,5-bisphosphate.

The basement membrane (BM), a specialized sheet of the extracellular matrix contacting the basal side of epithelial tissues, plays an important role in the control of the polarized structure of epithelial cells. However, little is known about how BM proteins themselves achieve a polarized distribution. Here, we identify phosphatidylinositol 4,5-bisphosphate (PIP2) as a critical regulator of the polarized secretion of BM proteins. A decrease of PIP2 levels, in particular through mutations in Phosphatidylinositol synthase (Pis) and other members of the phosphoinositide pathway, leads to the aberrant accumulation of BM components at the apical side of the cell without primarily affecting the distribution of apical and basolateral polarity proteins. In addition, PIP2 controls the apical and lateral localization of Crag (Calmodulin-binding protein related to a Rab3 GDP/GTP exchange protein), a factor specifically required to prevent aberrant apical secretion of BM. We propose that PIP2, through the control of Crag's subcellular localization, restricts the secretion of BM proteins to the basal side.

Phosphatidylinositol synthase activity from plant membrane fractions and from E. coli-expressed recombinant peptides.

Phosphatidylinositol (PtdIns) synthase is a lipid-synthesizing enzyme responsible for the synthesis of the phospholipid, PtdIns. Its enzymatic properties have been studied in in vitro assays using either membrane-enriched fractions or the purified protein in reconstituted lipid vesicles as a source of enzyme. More recently the specificities in terms of substrate preferences have also been studied using the recombinant protein expressed in Escherichia coli. This chapter deals with the purification of membranes as a source of PtdIns synthase before focusing on the in vitro assays of the enzymatic activities of the protein and, briefly, on the analysis of the product.

Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

Molecular interactions of natural and synthetic steroids in female hamsters' flank organs.

BACKGROUND: The initial step of steroidal action on target cells is gene activation; therefore, the quantification of mRNA is a direct method for comparing the role of different steroids in the skin. OBJECTIVE: This study demonstrated the role of several steroids on the mRNA expression encoding for different enzymes involved in the lipid metabolism in hamsters' flank organs, which are a pilosebaceous complex. METHODS: To determine the effect of treatments with testosterone (T) progesterone (P), levonorgestrel (LNG), 17α-p-chlorobenzoyloxy-6-chloropregn-4,6-diene-3,20-dione (5) and 17α-p-chlorobenzoyloxy-4,6-pregnadiene-3,20-dione (6); T and/or LNG; T and 5 or 6; P and/or 5 or 6 on the expression of mRNA encoding for lipid enzymes, the steroids were applied to the glands; later, the mRNAs expression for the enzymes was determined by PCR. The binding of 5 and 6 to the progesterone receptor (PR) was also evaluated. RESULTS: Treatments with T, LNG, T+LNG, P, T+P, 5, T+5, T+6, P, P+5 and P+6 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), ß-hydroxy-ß-methylglutaryl-CoA synthase (HMG-CoA-S), ß-hydroxy-ß-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase as compared to the controls. However, squalene synthase was increased with all treatments except with T+5 and 6; 6 did not significantly increase the expression for GPAT or HMG-CoA-S, however it increased the concentration of HMG-CoA-R enzyme. 5 and 6 bind to the PR, thus indicating that the effect of these steroids on the mRNA expression could be the result of their binding. CONCLUSION: The lipid metabolism is regulated by several steroids thought different mechanism of action, in flank organs.

Overexpression of the phosphatidylinositol synthase gene from Zea mays in tobacco plants alters the membrane lipids composition and improves drought stress tolerance.

Phosphatidylinositol (PtdIns) is an important lipid because it serves as a key membrane constituent and is the precursor of the inositol-containing lipids that are found in all plants and animals. It is synthesized from cytidine-diphosphodiacylglycerol (CDP-DG) and myo-inositol by PtdIns synthase (PIS). We have previously reported that two putative PIS genes from maize (Zea mays L.), ZmPIS and ZmPIS2, are transcriptionally up-regulated in response to drought (Sui et al., Gene, 426:47-56, 2008). In this work, we report on the characterization of ZmPIS in vitro and in vivo. The ZmPIS gene successfully complemented the yeast pis mutant BY4743, and the determination of PIS activity in the yeast strain further confirmed the enzymatic function of ZmPIS. An ESI-MS/MS-based lipid profiling approach was used to identify and quantify the lipid species in transgenic and wild-type tobacco plants before and after drought treatment. The results show that the overexpression of ZmPIS significantly increases lipid levels in tobacco leaves under drought stress compared to those of wild-type tobacco, which correlated well with the increased drought tolerance of the transgenic plants. Further analysis showed that, under drought stress conditions, ZmPIS overexpressors were found to exhibit increased membrane integrity, thereby enabling the retention of more solutes and water compared with the wild-type and the vector control transgenic lines. Our findings give us new insights into the role of the ZmPIS gene in the response of maize to drought/osmotic stress and the mechanisms by which plants adapt to drought stress.

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum.

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

Phosphatidylinositol synthase is required for lens structural integrity and photoreceptor cell survival in the zebrafish eye.

The zebrafish lens opaque (lop) mutant was previously isolated in a genetic screen and shown to lack rod and cone photoreceptors and exhibit lens opacity, or cataract, at 7 days post-fertilization (dpf). In this manuscript, we provide four different lines of evidence demonstrating that the lop phenotype results from a defect in the cdipt (phosphatidylinositol (PI) synthase; CDP-diacylglycerol-inositol 3-phosphatidyltransferase) gene. First, DNA sequence analysis revealed that the lop mutant contained a missense mutation in the lop open reading frame, which yields a nonconservative amino acid substitution (Ser-111-Cys) within the PI synthase catalytic domain. Second, morpholino-mediated knockdown of the cdipt-encoded PI synthase protein phenocopied the cdipt(lop/lop) mutant, with abnormal lens epithelial and secondary fiber cell morphologies and reduced numbers of photoreceptors. Third, microinjection of in vitro transcribed, wild-type cdipt mRNA into 1-4 cell stage cdipt(lop/lop) embryos significantly reduced the percentage of larvae displaying lens opacity at 7 dpf. Fourth, a cdipt retroviral-insertion allele, cdipt(hi559), exhibited similar lens and retinal abnormalities and failed to complement the cdipt(lop) mutant phenotype. To determine the initial cellular defects associated with the cdipt mutant, we examined homozygous cdipt(hi559/hi559) mutants prior to gross lens opacification at 6 dpf. The cdipt(hi559/hi559) mutants first exhibited photoreceptor layer disruption and photoreceptor cell death at 3 and 4 dpf, respectively, followed by lens dismorphogenesis by 5 dpf. RT-PCR revealed that the cdipt gene is maternally expressed and continues to be transcribed throughout development and into adulthood, in a wide variety of tissues. Using an anti-zebrafish PI synthase polyclonal antiserum, we localized the protein throughout the developing eye, including the photoreceptor layer and lens cortical secondary fiber cells. As expected, the polyclonal antiserum revealed that the PI synthase protein was reduced in amount in both the cdipt(lop/lop) and cdipt(hi559/hi559) mutants. Furthermore, we used a heterologous yeast phenotypic complementation assay to confirm that the wild-type zebrafish cdipt allele encodes functional PI synthase activity. Taken together, the cdipt-encoded PI synthase is required for survival of photoreceptor cells and lens epithelial and secondary cortical fiber cells. These zebrafish cdipt alleles represent excellent in vivo genetic tools to study the role of phosphatidylinositol and its phosphorylated derivatives in lens and photoreceptor development and maintenance.

Lack of de novo phosphatidylinositol synthesis leads to endoplasmic reticulum stress and hepatic steatosis in cdipt-deficient zebrafish.

UNLABELLED: Hepatic steatosis is the initial stage of nonalcoholic fatty liver disease (NAFLD) and may predispose to more severe hepatic disease, including hepatocellular carcinoma. Endoplasmic reticulum (ER) stress has been recently implicated as a novel mechanism that may lead to NAFLD, although the genetic factors invoking ER stress are largely unknown. During a screen for liver defects from a zebrafish insertional mutant library, we isolated the mutant cdipthi559Tg/+ (hi559). CDIPT is known to play an indispensable role in phosphatidylinositol (PtdIns) synthesis. Here we show that cdipt is expressed in the developing liver, and its disruption in hi559 mutants abrogates de novo PtdIns synthesis, resulting in hepatomegaly at 5 days postfertilization. The hi559 hepatocytes display features of NAFLD, including macrovesicular steatosis, ballooning, and necroapoptosis. Gene set enrichment of microarray profiling revealed significant enrichment of endoplasmic reticulum stress response (ERSR) genes in hi559 mutants. ER stress markers, including atf6, hspa5, calr, and xbp1, are selectively up-regulated in the mutant liver. The hi559 expression profile showed significant overlap with that of mammalian hepatic ER stress and NAFLD. Ultrastructurally, the hi559 hepatocytes display marked disruption of ER architecture with hallmarks of chronic unresolved ER stress. Induction of ER stress by tunicamycin in wild-type larvae results in a fatty liver similar to hi559, suggesting that ER stress could be a fundamental mechanism contributing to hepatic steatosis. CONCLUSION: cdipt-deficient zebrafish exhibit hepatic ER stress and NAFLD pathologies, implicating a novel link between PtdIns, ER stress, and steatosis. The tractability of hi559 mutant provides a valuable tool to dissect ERSR components, their contribution to molecular pathogenesis, and evaluation of novel therapeutics of NAFLD.

A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria.

For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely low compared with the PI synthase activity of yeast. When CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate were incubated with the cell wall components of Mycobacterium smegmatis, both phosphatidylinositol phosphate (PIP) and PI were formed, as identified by fast atom bombardment-mass spectrometry and thin-layer chromatography. PI was formed from PIP by incubation with the cell wall components. Thus, mycobacterial PI was synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via PIP, which was dephosphorylated to PI. The gene-encoding PIP synthase from four species of mycobacteria was cloned and expressed in Escherichia coli, and PIP synthase activity was confirmed. A very low, but significant level of free [(3)H]inositol was incorporated into PI in mycobacterial cell wall preparations, but not in recombinant E. coli cell homogenates. This activity could be explained by the presence of two minor PI metabolic pathways: PI/inositol exchange reaction and phosphorylation of inositol by ATP prior to entering the PIP synthase pathway.

Clinical significance of phosphatidyl inositol synthase overexpression in oral cancer.

BACKGROUND: We reported increased levels of phosphatidyl inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. METHODS: Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. RESULTS: Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. CONCLUSION: Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco.

Alternative metabolic fates of phosphatidylinositol produced by phosphatidylinositol synthase isoforms in Arabidopsis thaliana.

PtdIns is an important precursor for inositol-containing lipids, including polyphosphoinositides, which have multiple essential functions in eukaryotic cells. It was previously proposed that different regulatory functions of inositol-containing lipids may be performed by independent lipid pools; however, it remains unclear how such subcellular pools are established and maintained. In the present paper, a previously uncharacterized Arabidopsis gene product with similarity to the known Arabidopsis PIS (PtdIns synthase), PIS1, is shown to be an active enzyme, PIS2, capable of producing PtdIns in vitro. PIS1 and PIS2 diverged slightly in substrate preferences for CDP-DAG [cytidinediphospho-DAG (diacylglycerol)] species differing in fatty acid composition, PIS2 preferring unsaturated substrates in vitro. Transient expression of fluorescently tagged PIS1 or PIS2 in onion epidermal cells indicates localization of both enzymes in the ER (endoplasmic reticulum) and, possibly, Golgi, as was reported previously for fungal and mammalian homologues. Constitutive ectopic overexpression of PIS1 or PIS2 in Arabidopsis plants resulted in elevated levels of PtdIns in leaves. PIS2-overexpressors additionally exhibited significantly elevated levels of PtdIns(4)P and PtdIns(4,5)P(2), whereas polyphosphoinositides were not elevated in plants overexpressing PIS1. In contrast, PIS1-overexpressors contained significantly elevated levels of DAG and PtdEtn (phosphatidylethanolamine), an effect not observed in plants overexpressing PIS2. Biochemical analysis of transgenic plants with regards to fatty acids associated with relevant lipids indicates that lipids increasing with PIS1 overexpression were enriched in saturated or monounsaturated fatty acids, whereas lipids increasing with PIS2 overexpression, including polyphosphoinositides, contained more unsaturated fatty acids. The results indicate that PtdIns populations originating from different PIS isoforms may enter alternative routes of metabolic conversion, possibly based on specificity and immediate metabolic context of the biosynthetic enzymes.