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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.

Maladaptive impulsivity manifests in a variety of disorders, including attention-deficit hyperactivity disorder (ADHD), depression, and substance use disorder. However, the etiological mechanisms of impulsivity remain poorly understood. In the present study, we used in-vivo proton magnetic resonance spectroscopy (1H-MRS) to investigate neurometabolite content in the prefrontal cortex (PFC) and striatum of rats exhibiting low- versus high-impulsive (LI, HI) behavior on a visual attentional task. We validated our 1H-MRS findings using regionally resolved ex-vivo mass spectroscopy, transcriptomics, and site-directed RNA interference in the ventromedial PFC. We report a significant reduction in myoinositol levels in the PFC but not the striatum of HI rats compared with LI rats. Reduced myoinositol content was localized to the infralimbic (IL) cortex, where significant reductions in transcript levels of key proteins involved in the synthesis and recycling of myoinositol (IMPase1) were also present. Knockdown of IMPase1in the IL cortex increased impulsivity in nonimpulsive rats when the demand on inhibitory response control was increased. We conclude that diminished myoinositol levels in ventromedial PFC causally mediate a specific form of impulsivity linked to vulnerability for stimulant addiction in rodents. Myoinositol and related signaling substrates may thus offer novel opportunities for treating neuropsychiatric disorders comorbid with impulsive symptomology.

Phosphatidylinositol (PtdIns) serves as an integral component of eukaryotic membranes; however, its biosynthesis in apicomplexan parasites remains poorly understood. Here we show that Toxoplasma gondii—a common intracellular pathogen of humans and animals—can import and co-utilize myo-inositol with the endogenous CDP-diacylglycerol to synthesize PtdIns. Equally, the parasite harbors a functional PtdIns synthase (PIS) containing a catalytically-vital CDP-diacylglycerol phosphotransferase motif in the Golgi apparatus. Auxin-induced depletion of PIS abrogated the lytic cycle of T. gondii in human cells due to defects in cell division, gliding motility, invasion, and egress. Isotope labeling of the PIS mutant in conjunction with lipidomics demonstrated de novo synthesis of specific PtdIns species, while revealing the salvage of other lipid species from the host cell. Not least, the mutant showed decline in phosphatidylthreonine, and elevation of selected phosphatidylserine and phosphatidylglycerol species, indicating a rerouting of CDP-diacylglycerol and homeostatic inter-regulation of anionic phospholipids upon knockdown of PIS. In conclusion, strategic allocation of own and host-derived PtdIns species to gratify its metabolic demand features as a notable adaptive trait of T. gondii. Conceivably, the dependence of T. gondii on de novo lipid synthesis and scavenging can be exploited to develop new anti-infectives., Ren et al. investigated how the obligate intracellular pathogen Toxoplasma gondii satisfies its requirement of phosphatidylinositol (PtdIns). They show that this parasite can synthesize certain PtdIns species de novo and salvages others from human host cells. Endogenous production of PtdIns in the Golgi complex is critical for the acute infection, and its loss perturbs homeostasis of selected anionic phospholipids.

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., Author summary Phosphatidylserine (PS), a membrane phospholipid synthesized in the endoplasmic reticulum (ER) by the enzyme phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through different paths. The cellular functions of PS at different places in the cell and the mechanisms that coordinate the different PS transport paths are not fully understood. Here, we identified that PSS regulates cell growth, lipid storage and mitochondrial function in the fruit fly larval salivary gland. We showed that loss of pss function has three effects: (1) reduced levels of PS lead to reduced levels of plasma membrane Akt, a key component in the insulin pathway, which is important for cell growth; (2) it causes a shift from phospholipid synthesis to neutral lipid synthesis, which results in excess lipid accumulation; and (3) it reduces the level of mitochondrial PS, which impairs mitochondrial protein import and mitochondrial morphology. We also found that reducing the transport of PS from the ER to PM partially rescues the mitochondrial defects caused by loss of pss function. Together, our results reveal that PSS regulates cellular homeostasis through distinct metabolic changes, and uncover a balance between different PS transport pathways.

Autophagosomes engulf a variety of targets, from a portion of cytosol to large organelles, by regulating the size of the autophagosomal membrane. Yamamoto et al. identify ERdj8, a novel ER membrane protein that affects the size of autophagosomes., In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.

Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 Å), in complex with Mn2+ and citrate (1.9 Å), and with the CDP-DAG substrate (1.8 Å). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism—including a substrate-induced carboxylate shift—for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life., Kristīne Grāve et al. report the crystal structure of phosphatidylinositol phosphate synthase from Mycobacterium tuberculosis in its apo-state, in complex with Mn2+ and citrate, and bound to its CDP-DAG substrate. The structures provide atomic details of substrate binding and the dynamics of the catalytic metal site.

Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister–daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.

Phosphoinositides (PIPs) and their regulatory enzymes are key players in many cellular processes and are required for aspects of vertebrate development. Dysregulated PIP metabolism has been implicated in several human diseases, including a subset of skeletal myopathies that feature structural defects in the triad. The role of PIPs in skeletal muscle formation, and particularly triad biogenesis, has yet to be determined. CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) catalyzes the formation of phosphatidylinositol, which is the base of all PIP species. Loss of CDIPT should, in theory, result in the failure to produce PIPs, and thus provide a strategy for establishing the requirement for PIPs during embryogenesis. In this study, we generatedcdiptmutant zebrafish and determined the impact on skeletal myogenesis. Analysis ofcdiptmutant muscle revealed no apparent global effect on early muscle development. However, small but significant defects were observed in triad size, with T-tubule area, inter terminal cisternae distance and gap width being smaller incdiptmutants. This was associated with a decrease in motor performance. Overall, these data suggest that myogenesis in zebrafish does not requirede novoPIP synthesis but does implicate a role for CDIPT in triad formation.

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.

Cotton fibers are the most important natural raw material used in textile industries world-wide. Fiber length, strength, and fineness are the three major traits which determine the quality and economic value of cotton. It is known that exogenous application of phosphatidylinositols (PtdIns), important structural phospholipids, can promote cotton fiber elongation. Here, we sought to increase the in planta production of PtdIns to improve fiber traits. Transgenic cotton plants were generated in which the expression of a cotton phosphatidylinositol synthase gene (i.e., GhPIS) was controlled by the fiber-specific SCFP promoter element, resulting in the specific up-regulation of GhPIS during cotton fiber development. We demonstrate that PtdIns content was significantly enhanced in transgenic cotton fibers and the elevated level of PtdIns stimulated the expression of genes involved in PtdIns phosphorylation as well as promoting lignin/lignin-like phenolic biosynthesis. Fiber length, strength and fineness were also improved in the transgenic plants as compared to the wild-type cotton, with no loss in overall fiber yield. Our data indicate that fiber-specific up-regulation of PtdIns synthesis is a promising strategy for cotton fiber quality improvement.

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.

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., This work has been supported by grant number BFU2015-64440-P from the Spanish Ministerio de Economía, Industria y Competitividad and by FEDER. L.C. is supported by a fellowship from the Spanish Ministerio de Educación Cultura y Deporte. J.B. and E.L.E. were supported by the Academy of Finland.

In Eukarya, phosphatidylinositol (PI) is biosynthesized from CDP-diacylglycerol (CDP-DAG) and inositol. In Archaea and Bacteria, on the other hand, we found a novel inositol phospholipid biosynthetic pathway. The precursors, inositol 1-phosphate, CDP-archaeol (CDP-ArOH), and CDP-DAG, form archaetidylinositol phosphate (AIP) and phosphatidylinositol phosphate (PIP) as intermediates. These intermediates are dephosphorylated to synthesize archaetidylinositol (AI) and PI. To date, the activities of the key enzymes (AIP synthase, PIP synthase) have been confirmed in only three genera (two archaeal genera, Methanothermobacter and Pyrococcus, and one bacterial genus, Mycobacterium). In the present study, we demonstrated that this novel biosynthetic pathway is universal in both Archaea and Bacteria, which contain inositol phospholipid, and elucidate the specificity of PIP synthase and AIP synthase for lipid substrates. PIP and AIP synthase activity were confirmed in all recombinant cells transformed with the respective gene constructs for four bacterial species (Streptomyces avermitilis, Propionibacterium acnes, Corynebacterium glutamicum, and Rhodococcus equi) and two archaeal species (Aeropyrum pernix and Sulfolobus solfataricus). Inositol was not incorporated. CDP-ArOH was used as the substrate for PIP synthase in Bacteria, and CDP-DAG was used as the substrate for AIP synthase in Archaea, despite their fundamentally different structures. PI synthase activity was observed in two eukaryotic species, Saccharomyces cerevisiae and Homo sapiens; however, inositol 1-phosphate was not incorporated. In Eukarya, the only pathway converts free inositol and CDP-DAG directly into PI. Phylogenic analysis of PIP synthase, AIP synthase, and PI synthase revealed that they are closely related enzymes.

Glycerophospholipids are the principal fabric of cellular membranes. The pathways by which these lipids are synthesized were elucidated mainly through the work of Kennedy and colleagues in the late 1950s and early 1960s. Subsequently, attention turned to cell biological aspects of lipids: Where in the cell are lipids synthesized? How are lipids integrated into membranes to form a bilayer? How are they sorted and transported from their site of synthesis to other cellular destinations? These topics, collectively termed 'lipid topogenesis', were the subject of a review article in 1981 by Bell, Ballas and Coleman. We now assess what has been learned about early events of lipid topogenesis, i.e. "lipid synthesis, the integration of lipids into membranes, and lipid translocation across membranes", in the 35 years since the publication of this important review. We highlight the recent elucidation of the X-ray structures of key membrane enzymes of glycerophospholipid synthesis, progress on identifying lipid scramblase proteins needed to equilibrate lipids across membranes, and new complexities in the subcellular location and membrane topology of phosphatidylinositol synthesis revealed through a comparison of two unicellular model eukaryotes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

We have identified Rab10 as an ER-specific Rab GTPase that regulates ER structure and dynamics. We show that Rab10 localizes to the ER and to dynamic ER-associated structures that track along microtubules and mark the position of new ER tubule growth. Rab10 depletion or expression of a Rab10 GDP-locked mutant alters ER morphology, resulting in fewer ER tubules. We demonstrate that this defect is due to a reduced ability of dynamic ER tubules to grow out and successfully fuse with adjacent ER. Consistent with this function, Rab10 partitions to dynamic ER-associated domains found at the leading edge of almost half of all dynamic ER tubules. Interestingly, this Rab10 domain is highly enriched with at least two ER enzymes that regulate phospholipid synthesis, phosphatidylinositol synthase (PIS) and CEPT1. Both the formation and function of this Rab10/PIS/CEPT1 dynamic domain are inhibited by expression of a GDP-locked Rab10 mutant. Together, these data demonstrate that Rab10 regulates ER dynamics and further suggest that these dynamics could be coupled to phospholipid synthesis.

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), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-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.

In our recent paper, we biochemically analyzed autophagosome-related membranes at the initiation stage of macroautophagy/autophagy using atg knockout (KO) cells and demonstrated that the ULK complex is recruited to 2 distinct membranes: the ER membrane and ATG9A-positive autophagosome precursors. We have also identified phosphatidylinositol synthase (PIS)-enriched ER subdomains as the initiation site of autophagosome formation. Based on these findings, we propose that the ULK complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.

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.

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. (HEPATOLOGY 2011;)

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.

The last several years have been witness to significant developments in understanding transcriptional regulation of the yeast phospholipid structural genes. The response of most phospholipid structural genes to inositol is now understood on a mechanistic level. The roles of specific activators and repressors are also well established. The knowledge of specific regulatory factors that bind the promoters of phospholipid structural genes serves as a foundation for understanding the role of chromatin modification complexes. Collectively, these findings present a complex picture for transcriptional regulation of the phospholipid biosynthetic genes. The INO1 gene is an ideal example of the complexity of transcriptional control and continues to serve as a model for studying transcription in general. Furthermore, transcription of the regulatory genes is also subject to complex and essential regulation. In addition, databases resulting from a plethora of genome-wide studies have identified regulatory signals that control one of the essential phospholipid biosynthetic genes, PIS1. These databases also provide significant clues for other regulatory signals that may affect phospholipid biosynthesis. Here, we have tried to present a complete summary of the transcription factors and mechanisms that regulate the phospholipid biosynthetic genes.

Phosphatidylinositol (PI) is a versatile lipid that not only serves as a structural component of cellular membranes, but also plays important roles in membrane anchorage of proteins and in signal transduction through distinct phosphorylated derivatives of the inositol head group. PI is synthesised by PI synthase from CDP-diacylglycerol and myo-inositol. The enzymatic activity in Plasmodium falciparum and P. knowlesi has previously been characterised at the biochemical level. Here we characterise the PI synthase gene of P. falciparum and P. knowlesi. The cDNA sequence identified a highly spliced gene consisting of nine exons and encoding a protein of 209 and 207 amino acids, respectively. High sequence conservation enabled the prediction of the PI synthase genes of P. berghei, P. chabaudi and P. vivax. All Plasmodium PI synthase proteins appear to be highly hydrophobic, although no consensus for the number and location of distinct transmembrane domains could be detected. The P. falciparum PI synthase (PfPIS) gene successfully complemented a Saccharomyces cerevisiae PIS1 deletion mutant, demonstrating its enzymatic function. Complementation efficiency was dramatically improved when hybrid constructs between N-terminal S. cerevisiae and C-terminal P. falciparum sequences were used. Determination of in vitro PIS activities of complemented yeast strains confirmed the enzymatic function of the Plasmodium protein.

Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.

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., CDP-alcohol phosphotransferases (CDP-APs) are critical for the biosynthesis of glycerophospholipids. Here, Clarke et al. present the first structure of an enzymatically active CDP-AP in the presence of a bound lipid substrate and propose a mechanism for substrate binding and catalysis.

Phosphatidylinositol is a metabolic precursor of phosphoinositides and soluble inositol phosphates. Both sets of molecules represent versatile intracellular chemical signals in eukaryotes. While much effort has been invested in understanding the enzymes that produce and consume these molecules, central aspects for how phosphoinositide production is controlled and functionally partitioned remain unresolved and largely unappreciated. It is in this regard that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as central regulators of the functional channeling of phosphoinositide pools produced on demand for specific signaling purposes. The physiological significance of these proteins is amply demonstrated by the consequences that accompany deficits in individual PITPs. Although the biological problem is fascinating, and of direct relevance to disease, PITPs remain largely uncharacterized. Herein, we discuss our perspectives regarding what is known about how PITPs work as molecules, and highlight progress in our understanding of how PITPs are integrated into cellular physiology. This article is part of a Special Issue entitled Phosphoinositides.

The effects of resveratrol (trans-3,4′,5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) were monitored in blood platelets prelabelled with [32P]Pi. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of 32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P2 available for signalling in these cells.

Phosphatidylinositol (PtdIns) synthase 1 from the plant Arabidopsis thaliana has been expressed in Escherichia coli in order to study the synthetic capacities of the enzyme. Analysis of the total fatty acid content of the bacteria shows that PtdIns synthase activity does not have a profound effect on the proportions of the different fatty acids produced, even if the presence of an extra acidic phospholipid leads to a global reduction of the lipid content. A closer analysis carried out on individual phospholipids reveals a global fatty acid composition almost unchanged in the two major bacterial lipids phosphatidylethanolamine (PtdEtn) and phosphatidylglycerol (PtdGro). Phosphatidylinositol has a very unusual composition that shows the ability of the plant enzyme to use CDP-diacylglycerol molecular species absent from plants. We identified the various PtdIns molecular species. They represent a pool of the major molecular species of PtdEtn and PtdGro. These results, together with the determination of the apparent affinity constants of AtPIS1 for myo-inositol and CDP-diacylglycerol, allow us to discuss some of the constraints of PtdIns synthesis in plants in terms of specificity, which will depend on the subcellular localization of the protein.

The anti-anginal drug trimetazidine (TMZ) has been shown to increase the synthesis of phospholipids in ventricular myocytes, including phosphatidyl-inositol (PI). This study focused on the consequences of increasing PI metabolism on alpha-adrenergic signaling pathway in cultured rat cardiomyocytes. In the cells treated with TMZ, the synthesis of PI from inositol was largely increased as compared with the control (+55% in 60 min). The stimulation of alpha-adrenergic receptors by phenylephrine (PE) induced a dose-dependent production of inositide phosphates (IPs) by phospholipase C (PLC) activation. However, the amount of available IPs was significantly lower in TMZ-treated cells, in a dose-dependent manner. This effect was observed in the presence and absence of the IP1-phosphatase inhibitor LiCl. The in vitro determination of PLC activity revealed that this effect could not be attributed to the direct inhibition of the enzyme by TMZ. The TMZ-induced reduction of IPs in the PE-stimulated cardiomyocytes should be attributed to the increase of inositol recycling and incorporation in membrane structures, elicited by increased phospholipid synthesis. The consequences of this reduction in IPs availability were investigated on the cardiomyocyte hypertrophy induced by alpha-adrenergic chronic stimulation. Acute stimulation with PE increased protein synthesis (+50%), but this increase was largely prevented by TMZ. In conclusion, TMZ reduces cell available IPs, by accelerating their recycling in membranes as PI. This effect results in a cytoprotection in the pathological process of hypertrophy elicited by chronic alpha-adrenergic stimulation.

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)‐diacylglycerol (DG): myo‐inositol 3–phos–phatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP‐DG and myo‐inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH‐PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH‐PIS cells showed an increase in PI synthesis in vitro and in vivo, as well as increased cellular levels of PI–4,5–P2 and PI–3,4,5–P3. They also displayed a decrease in their doubling tune and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum‐stimulated quiescent NIH3T3 cells. Moreover, PIS Overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.

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.

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)P2 production 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)P2 hydrolysis and PtdIns3P production are essential for normal cytokinesis abscission. Finally, emerging functions of PtdIns3P and likely PtdIns(4,5)P2 have 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.

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.

The apicomplexan parasite Toxoplasma gondii has the ability to switch between a rapidly replicating tachyzoite and a slowly dividing encysted bradyzoite within its intermediate hosts such as humans or other warm-blooded vertebrates. It is likely that in vivo, the tachyzoites differentiate into encysted bradyzoites in response to the immune system attack during disease progression. As part of a developmental strategy and, in order to survive within infected hosts, T. gondii tachyzoites undergo profound metabolic and morphological changes by differentiating into encysted bradyzoites. Bradyzoites are characterised by their resistance to both the immune system and chemotherapy. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms triggering the switch from tachyzoite to bradyzoite remain unknown. It is very important to elucidate these mechanisms since bradyzoites within tissue cysts are not only the source of infection transmitted from domestic animals to humans, but can also be converted into tachyzoites that are the cause of fatal toxoplasmic encephalitis in acquired immunodeficiency syndrome patients. In this review, I focus on recent efforts towards the characterisation of genes that encode several stage-specific isoenzymes. The picture emerging from these studies is that stage-specific expression of isoenyzmes having different biochemical properties accompanies the interconversion of tachyzoite into bradyzoite, and vice versa. It can be hypothesised that the difference found between these enzymatic activities may be instrumental in maintaining some major parasitic metabolisms such as glycolysis in pace with the stage-specific requirements of carbohydrate or polysaccharide biosynthesis.

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.

Human erythrocytes were loaded with myo-( 3 H)-inositol in the presence or absence of cytidine trisphosphate to investigate the synthesis of membrane phosphoinositides in the intact red cell. The addition of cytidylic nucleotides to the loading mixture yielded a four-fold increase in the ( 3 H)-labeling of the membranes. The ( 3 H)-labeling of phosphatidylinositol, phosphati- dylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was distinguished by two chromatographic techniques. Experi- ments performed on white ghosts demonstrated the presence of CDP-diacylglycerol synthase and phosphatidylinositol synthase. These results and those already reported allow to discuss a possible turnover of the inositol polar head. © Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS phosphoinositides / erythrocyte / myo-inositol / loading / membrane

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M . smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M . smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [ 3 H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [ 3 H]PI and NBD-PI yield by M . smegmatis . At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [ 3 H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K m of the enzyme toward inositol was shown to be 25 μM; Mg 2+ stimulated the enzyme to a greater degree than Mn 2+ . Structural analogs of myo -inositol, epi -inositol and scyllo -inositol and Zn 2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for anti-tuberculosis therapy.

The regulation of phosphatidylinositol synthesis was examined by cloning and expressing in COS-7 cells the human cDNAs encoding the two enzymes in the biosynthetic pathway. Human CDP-diacylglycerol synthetase (cds1) and phosphatidylinositol synthase (pis1) clones were identified in the human expressed sequence-tagged (EST) data base, and full-length cDNAs were obtained by library screening. The cds1 cDNA did not possess a recognizable mitochondrial import signal, and the activity of the expressed Cds1 protein was stimulated by nucleoside triphosphates in vitro, indicating that cds1 did not encode the mitochondrial-specific isozyme. There were two mRNA species (3.9 and 5.6 kilobases) detected on Northern blots hybridized with the cds1 probe that were expressed at distinctly different levels in various human tissues. Consistent with the presence of the two mRNAs, a cDNA predicted to encode a second human CDP-diacylglycerol synthetase (cds2) was also uncovered in the EST data base. In contrast to the two cds mRNAs, a single, 2.1-kilobase pis1 mRNA was uniformly expressed in all human tissues examined. Expression of the pis1 gene led to the overproduction of both phosphatidylinositol synthase and phosphatidylinositol:inositol exchange reactions, indicating that the Pis1 polypeptide catalyzed both of these activities. Phosphatase treatment of cell extracts abolished the CMP-independent phosphatidylinositol:inositol exchange reaction, and exchange activity was completely restored by the addition of CMP. Overexpression of cds1 or pis1 alone or in combination did not enhance the rate of phosphatidylinositol biosynthesis. Also, overexpression did not result in a significant proportional increase in the cellular levels of CDP-diacylglycerol or phosphatidylinositol. These data illustrate that the levels of Cds1 and Pis1 protein expression are not critical determinants of cellular PtdIns content and argue against a determining role for the activity of either of these enzymes in the regulation of PtdIns biosynthesis.

Phosphatidylinositol synthase (CDP-diacylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11) is a 24-kDa membrane-bound enzyme. It is present in all mammalian cells and is localized predominantly to the endoplasmic reticulum. The enzyme performs the last step in the de novo biosynthesis of the phospholipid phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to form the products phosphatidylinositol and CMP. Phosphatidylinositol, apart from being an essential membrane phospholipid, is involved in protein membrane anchoring and is the precursor for the second messengers inositol-tri-phosphate and diacylglycerol.

Amine-containing phospholipid synthesis in Saccharomyces cerevisiae starts with the conversion of CDP-diacylglycerol (CDP-DAG) and serine to phosphatidylserine (PS), whereas phosphatidylinositol (PI) is formed from CDP-DAG and inositol (derived from inositol 1-phosphate). In this study the regulation of PS synthase (encoded by CHO1/PSS), PI synthase (encoded by PIS1), and inositol 1-phosphate synthase (encoded by INO1) activities by the in vivo level of CDP-DAG synthase activity (encoded by CDS1) is described. Reduction in the level of CDP-DAG synthase activity from 10-fold over wild type levels to 10% of wild type levels results in a 7-fold increase in PS synthase activity, which follows a similar change in the CHO1/PSS mRNA level. INO1 mRNA also increases but only after CDP-DAG synthase activity falls below the wild type level. PI synthase activity follows the decrease of the CDP-DAG synthase activity, but there is no parallel change in the level of PIS1 mRNA. These changes in CHO1/PSS and INO1 mRNA levels are mediated by a mechanism not dependent on changes in the expression of the INO2-OPI1 regulatory genes. CDS1 expression is repressed in concert with INO2 expression in response to inositol.

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.

The CDIPT is crucial to the fatty acid metabolic pathway, intracellular signal transduction and energy metabolism in eukaryotic cells. We detected three SNPs at 3'-untranslated regions (UTR), named 3'-UTR_108 A > G, 3'-UTR_448 G > A and 3'-UTR_477 C > G, of the CDIPT gene in 618 Qinchuan cattle using PCR-RFLP and DNA sequencing methods. At each of the three SNPs, we found three genotypes named as follows: AA, AB, BB (3'-UTR_108 A > G), CC, CD, DD (3'-UTR_448 G > A) and EE, EF, FF (3'-UTR_477 C > G.). Based on association analysis of these SNPs with ultrasound measurement traits, individuals of genotype BB had a significantly larger loin muscle area than genotype AA. Individuals of genotype CC had significantly thicker back fat than individuals of genotype DD. Individuals of genotype EE also had significantly thicker back fat than did individuals of genotype FF. We conclude that these SNPs of the CDIPT gene could be used as molecular markers for selecting and breeding beef cattle with superior body traits, depending on breeding goals.

Flank organs are an androgen-dependent pilosebaceous complex present in male and female hamsters. These organs have been used for the evaluation of antiandrogenic drugs, which could be used for the treatment of androgen-dependent afflictions. This study demonstrated the role of four different steroidal carbamates 7-10 in the expression of mRNAs coding for different enzymes involved in the lipid metabolism in flank organs. To determine the biological effects of compounds 7-10 on the expression of mRNA coding for lipid enzymes, steroids 7-10, testosterone (T), progesterone (P), and/or 7-10 were applied on the flank organs. Later, the mRNA expression for the enzymes was determined by polymerase chain reaction. The binding of 8 and 9 to the progesterone receptor (PR) as well as their effects on the activity of 5α-reductase were also evaluated. Treatments with T, P, and 7-10 increased the mRNA expression for glycerol 3-phosphate acyl transferase (GPAT), β-hydroxy-β-methylglutaryl-CoA synthase (HMG-CoA-S), β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA-R), phosphatidylinositol synthase (PI-S), and squalene-synthase (SQ-S). However, the combined treatments with P + 7-10 decreased the expression of GPAT, HMG-CoA-S, and HMG-CoA-R. Expression of mRNA for all enzymes was variable under treatment with T + 7-10. Data showed that these carbamates did not bind to the PR, but inhibited the activity of 5α-reductase. Carbamates 7-10 changed the mRNA expression model induced by T and P in flank organs.

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol: 3-phosphatidyltransferase, EC 2.7.8.11) catalyzes the formation of phosphatidylinositol and CMP from CDP-diacyl-glycerol and myo-inositol. We have cloned a phosphatidylinositol synthase cDNA from rat brain by functional complementation of the yeast pis mutation, which is defective in phosphatidylinositol synthase. The deduced protein comprised 213 amino acids with a calculated molecular mass of 23 613 Da. The predicted protein sequence is highly homologous to the previously determined yeast phosphatidylinositol synthase sequence. The cDNA hybridized to a 1.7-kb mRNA that was abundantly expressed in rat brain and kidney.

PI-synthase selectivity from etiolated maize coleoptiles was studied either associated with the microsomal membranes or after solubilization by CHAPS and prepurification on a DEAE-trisacryl M column. When maize microsomes were incubated with [3H]inositol without any exogenous CMM-PA, the most heavily labelled molecular species were 16:0/18:2-PI (77%), 16:0/18:3- plus 18:2/18:2-PI (15%), 16:0/18:1-PI (4%) and 18:0/18:2-PI (4%). Addition to the incubation medium of up to 300 μM 16:0/16:0-CMP-PA unexpectedly resulted in the formation of very little labelled 16:0/16:0-PI. When the solubilized fraction from microsomes was incubated with [3H]inositol in absence of 16:0/16:0-CPM-PA, the same PI molecular species as above were synthesized. However, with increasing concentrations of 16:0/16:0-CMP-PA in the medium, increasing amounts of labelled 16:0/16:0-PI appeared as well. With prepurified PI-synthase eluted from a DEAE column, endogenous CMP-PA was poorly utilized for PI biosynthesis whereas the exogenous 16:0/16:0-CPM-PA was used actively. With time, the endogenous CMP-PA was utilized first and the exogenous substrate was utilized, albeit, much more slowly. The results demonstrate that the selectivity displayed by PI-synthase towards various molecular species of CMP-PA depends on the integration of the enzyme in the membrane structure. Solubilization of the enzyme, i.e., inclusion of the protein in micelles with detergents and lipids, results in an apparent loss of the selectivity for CMP-PA.

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.

Membranes from human brain cortex (8-12 h post mortem) were labelled with [3H]inositol, in the presence of CMP, through the back reaction catalysed by PtdIns synthase. The enzyme incorporated [3H]inositol into phosphoinositides at a maximal rate of 419 pmol min-1 mg protein-1. In the absence of CMP, the labelling rate due to the PtdIns headgroup exchanging enzyme was 36 pmol min-1 mg protein-1. Human brain PtdIns synthase showed Kmapp values of 0.49 mM and 18 microM for inositol and CMP, respectively. In the presence of ATP, [3H]polyphosphoinositides formed after [3H]PtdIns were hydrolysed by phospholipase C in a GTP gamma S and neurotransmitter receptor agonist-dependent manner. Production of 3H-inositol phosphates as stimulated by GTP gamma S (350% of basal) was increased by the muscarinic agonists carbachol and oxotremorine-M (600% of basal) and by serotonin (485% of basal). The relative potencies of carbachol and oxotremorine-M were consistent with an action at muscarinic receptors. These results show that coupling between muscarinic and serotonin receptors and phospholipase C is preserved in membranes from post mortem human brain cortex and validate the use of a method involving direct [3H]inositol labelling of a membrane fraction to study the functional state of phospholipase C-coupled receptors in human brain samples.