Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg 2؉ -independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1⌬ mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1⌬ mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg 2؉ -independent PA phosphatase activity in S. cerevisiae. Diacylglycerol pyrophosphate (DGPP)1 is a novel phospholipid that contains a pyrophosphate group attached to diacylglycerol (DG) (Fig. 1) (1). DGPP has been found in a variety of plants (2, 3) and in the yeast Saccharomyces cerevisiae (4). This phospholipid is synthesized from phosphatidate (PA) and ATP via the reaction catalyzed by the membrane-associated enzyme PA kinase (1) and is dephosphorylated to PA via the reaction catalyzed by the membrane-associated enzyme DGPP phosphatase ( Fig. 1) (4). The amounts of DGPP in wild-type S. cerevisiae and in plants are barely detectable (3, 4). For example, DGPP accounts for only 0.18 mol % of the major phospholipids in S. cerevisiae (4). The low abundance of DGPP is reminiscent of lipid signaling molecules such as the inositol-containing phospholipids (5-9). Recent studies indicate that the metabolism of DGPP is involved in a novel lipid signaling pathway. DGPP accumulates in plant tissues upon G protein activation through the stimulation of PA kinase activity (3), and metabolic labeling studies with Catharanthus roseus cells have shown that DGPP is metabolized rapidly to PA and then to DG (10). It has been suggested that DGPP may function as a signaling molecule (3, 4). Alternatively, the formation of DGPP may serve to attenuate the signaling functions of PA (11,12).DGPP phosphatase activity has been identified in S. cerevisiae, C. roseus, Escherichia coli, rat liver, pig l...

Sphingolipid metabolism is implicated to play an important role in apoptosis. Here we show that dihydrosphingosine (DHS) and phytosphingosine (PHS), two major sphingoid bases of fungi, have potent fungicidal activity with remarkably high structural and stereochemical specificity against Aspergillus nidulans. In fact, only naturally occurring DHS and PHS are active. Further analysis revealed that DHS and PHS induce rapid DNA condensation independent of mitosis, large-scale DNA fragmentation, and exposure of phosphatidylserine, all common morphological features characteristic of apoptosis, suggesting that DHS and PHS induce apoptosis in A. nidulans. The finding that DNA fragmentation requires protein synthesis, which implies that an active process is involved, further supports this proposition. The induction of apoptosis by DHS and PHS is associated with the rapid accumulation of reactive oxygen species (ROS). However, ROS are not required for apoptosis induced by DHS and PHS, as scavenging of ROS by a free radical spin trap has no effect. We further demonstrate that apoptosis induced by DHS and PHS is independent of metacaspase function but requires mitochondrial function. Together, the results suggest that DHS and PHS induce a type of apoptosis in A. nidulans most similar to the caspase-independent apoptosis observed in mammalian systems. As A. nidulans is genetically tractable, this organism should be an ideal model system for dissecting sphingolipid signaling in apoptosis and, importantly, for further elucidating the molecular basis of caspase-independent apoptosis.In recent years, mounting evidence has indicated that sphingolipid metabolism is an important cell signaling system. Rapid and transient changes in sphingolipid metabolism are closely associated with a wide range of cellular activities, including the stress response, apoptosis, inflammation, cell cycle regulation, and cancer development (19,20,33). For instance, stress signals rapidly and transiently elevate the level of cellular ceramide; conversely, growth factors stimulate the rapid, transient generation of sphingosine-1-phosphate (S-1-P). Importantly, the treatment of cells with short-chain ceramide analogs or the expression of sphingomyelinases reproduces most of the effects of endogenous ceramide, particularly in the stress response and apoptosis (19,20,33). Furthermore, S-1-P, as a highaffinity ligand of the edg family of G-protein-coupled receptors, promotes cell survival and proliferation by antagonizing ceramide-mediated apoptosis (52, 61). Thus, ceramide, as an important regulatory component in the stress response and apoptosis, and S-1-P, involved in cell survival and proliferation, have attracted enormous scientific interest in recent years. In particular, a large body of evidence has now accumulated to implicate an important role of ceramide in the stress response and apoptosis. It is proposed that the relative levels of cellular ceramide and S-1-P determine whether cells undergo apoptosis or continue to proliferate (61).The sphingoid ...

Sphingolipids are major components of the plasma membrane of eukaryotic cells and were once thought of merely as structural components of the membrane. We have investigated effects of inhibiting sphingolipid biosynthesis, both in germinating spores and growing hyphae of Aspergillus nidulans. In germinating spores, genetic or pharmacological inactivation of inositol phosphorylceramide (IPC) synthase arrests the cell cycle in G 1 and also prevents polarized growth during spore germination. However, inactivation of IPC synthase not only eliminates sphingolipid biosynthesis but also leads to a marked accumulation of ceramide, its upstream intermediate. We therefore inactivated serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthesis pathway, to determine effects of inhibiting sphingolipid biosynthesis without an accumulation of ceramide. This inactivation also prevented polarized growth but did not affect nuclear division of germinating spores. To see if sphingolipid biosynthesis is required to maintain polarized growth, and not just to establish polarity, we inhibited sphingolipid biosynthesis in cells in which polarity was already established. This inhibition rapidly abolished normal cell polarity and promoted cell tip branching, which normally never occurs. Cell tip branching was closely associated with dramatic changes in the normally highly polarized actin cytoskeleton and found to be dependent on actin function. The results indicate that sphingolipids are essential for the establishment and maintenance of cell polarity via control of the actin cytoskeleton and that accumulation of ceramide is likely responsible for arresting the cell cycle in G 1 .

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

Phosphatidylinositol-3-kinase (PI3K), and its downstream effector Akt, or protein kinase B␣ (PKB␣), play a major regulatory role in control of apoptosis, proliferation, and angiogenesis. PI3K and Akt are amplified or overexpressed in a number of malignancies, including sarcomas, ovarian cancer, multiple myeloma, and melanoma. This pathway regulates production of the potent angiogenic factor vascular endothelial growth factor (VEGF), and protects tumor cells against both chemotherapy and reactive oxygen-induced apoptosis through phosphorylation of substrates such as apoptotic peptidaseactivating factor-1 (APAF-1), forkhead proteins, and caspase 9. Given its diverse actions, compounds that suppress the PI3K/Akt pathway have potential pharmacologic utility as angiogenesis inhibitors and antineoplastic agents. Using the SVR angiogenesis assay, a screen of natural products, we isolated the alkaloid solenopsin, and found that it is a potent angiogenesis inhibitor. We also found that solenopsin inhibits the PI3K signaling pathway in IntroductionThe serine/threonine kinase c-Akt-1, or protein kinase B␣ (PKB), is the cellular homolog of a transforming oncogene initially isolated from a lymphoma. Akt is a downstream target of phosphatidylinositol-3-kinase (PI3K), a family of at least 4 different enzymes, with the prototypical PI3K heterodimer consisting of a p85 (regulatory) and a p110 (catalytic) subunit. The PI3K/Akt pathway is involved in the regulation of diverse cellular functions including proliferation, cytoskeletal organization, survival, and malignant transformation. [1][2][3][4] Upon binding of PI3K products to its pleckstrin homology domain, Akt is translocated to the plasma membrane where it is activated by upstream phosphorylated kinases, including PI3K-dependent kinases 1 and 2 (PDK1 and PDK2) and mammalian target of rapamycin complex 2 (mTORC2). The PI3K/Akt pathway is stimulated by numerous receptor tyrosine kinases and oncogenes, including receptors for insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), ras, Her2/neu, and polyoma middle T oncogenes. [5][6][7][8][9][10] Because Akt plays a central role in regulating apoptosis, angiogenesis, and metabolism of cells, Akt is an attractive pharmacologic target for the treatment of cancer and inflammation. 11,12 Small-molecular-weight inhibitors of PI3K include LY 294002 and the fungal metabolite wortmannin, 13 as well as ether phospholipids, including perifosine, which has entered clinical trials. 14,15 Using the SVR angiogenesis assay, [16][17][18] we found that solenopsin A, 19,20 the primary alkaloid from the fire ant Solenopsis invicta, has antiangiogenic activity. We also discovered that solenopsin disrupted angiogenesis in vivo in embryonic zebrafish. In order to determine the mode of action, we examined the ability of solenopsin to inhibit a battery of cellular kinases and found that solenopsin A inhibited Akt relatively selectively in an ATP-competitive manner without affecting its u...

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

Acyl carrier protein synthase (AcpS) catalyzes the formation of holo‐ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3′5′‐ADP, a product of AcpS, at 2.0 and 1.9 Å resolution, respectively. The crystal structure reveals an α/β fold and shows that AcpS assembles as a tightly packed functional trimer, with a non‐crystallographic pseudo‐symmetric 3‐fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure‐based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.