Crystal structure of a lipin/Pah phosphatidic acid phosphatase.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

Purification of Lipin and Measurement of Phosphatidic Acid Phosphatase Activity from Liposomes.

The lipin family of enzymes are phosphatidic acid (PA) phosphatases responsible for converting PA to diacylglycerol (DAG). Lipins therefore occupy a central node in the synthesis of triacylglycerol (TAG) and phospholipids, and may play a role in regulating the levels of PA and DAG as signaling molecules. Some enzymatic assays used to measure PA phosphatase activities use detergents above their critical micelle concentration to present substrate; however, these methods do not represent the physiological membrane bilayers found in cells and these conditions can drastically alter phosphatase activities. Other assays use poorly defined mixtures of phosphatidylcholine (PC), PA, and high concentrations of BSA to present substrate. In this chapter, we describe methods for affinity purification of FLAG-tagged lipin proteins, and an alternative enzymatic assay using small unilamellar vesicles, also known as liposomes, to investigate specific activities of PA phosphatases. These activities are measured using an acidified Bligh-Dyer extraction to separate the water-soluble, radiolabeled, inorganic phosphate released during the assay from the chloroform-soluble PA.

Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei.

BACKGROUND: Arginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of proteins whose functions are modulated by arginine methylation await discovery in trypanosomes. Here, we employed a yeast two-hybrid screen using as bait the major T. brucei type I PRMT, TbPRMT1, to identify potential substrates of this enzyme. RESULTS: We identified a protein containing N-LIP and C-LIP domains that we term TbLpn. These domains are usually present in a family of proteins known as lipins, and involved in phospholipid biosynthesis and gene regulation. Far western and co-immunoprecipitation assays confirmed the TbPRMT1-TbLpn interaction. We also demonstrated that TbLpn is localized mainly to the cytosol, and is methylated in vivo. In addition, we showed that, similar to mammalian and yeast proteins with N-LIP and C-LIP domains, recombinant TbLpn exhibits phosphatidic acid phosphatase activity, and that two conserved aspartic acid residues present in the C-LIP domain are critical for its enzymatic activity. CONCLUSIONS: This study reports the characterization of a novel trypanosome protein and provides insight into its enzymatic activity and function in phospholipid biosynthesis. It also indicates that TbLpn functions may be modulated by arginine methylation.

Characterization of the yeast actin patch protein App1p phosphatidate phosphatase.

Yeast App1p is a phosphatidate phosphatase (PAP) that associates with endocytic proteins at cortical actin patches. App1p, which catalyzes the conversion of phosphatidate (PA) to diacylglycerol, is unique among Mg(2+)-dependent PAP enzymes in that its reaction is not involved with de novo lipid synthesis. Instead, App1p PAP is thought to play a role in endocytosis because its substrate and product facilitate membrane fission/fusion events and regulate enzymes that govern vesicular movement. App1p PAP was purified from yeast and characterized with respect to its enzymological, kinetic, and regulatory properties. Maximum PAP activity was dependent on Triton X-100 (20 mm), PA (2 mm), Mg(2+) (0.5 mm), and 2-mercaptoethanol (10 mm) at pH 7.5 and 30 °C. Analysis of surface dilution kinetics with Triton X-100/PA-mixed micelles yielded constants for surface binding (Ks(A) = 11 mm), interfacial PA binding (Km(B) = 4.2 mol %), and catalytic efficiency (Vmax = 557 µmol/min/mg). The activation energy, turnover number, and equilibrium constant were 16.5 kcal/mol, 406 s(-1), and 16.2, respectively. PAP activity was stimulated by anionic lipids (cardiolipin, phosphatidylglycerol, phosphatidylserine, and CDP-diacylglycerol) and inhibited by zwitterionic (phosphatidylcholine and phosphatidylethanolamine) and cationic (sphinganine) lipids, nucleotides (ATP and CTP), N-ethylmaleimide, propranolol, phenylglyoxal, and divalent cations (Ca(2+), Mn(2+), and Zn(2+)). App1p also utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA.

Simple screening method for improving membrane protein thermostability.

Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.

Cloning, expression, and characterization of a thermostable PAP2L2, a new member of the type-2 phosphatidic acid phosphatase family from Geobacillus toebii T-85.

Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding a PAP2-like protein, Gtb PAP2L2, which contains 212 amino acids and shows a limited homology to other known PAP2s, especially at conserved phosphatase motifs, and a similar six-transmembrane topology structure. This enzyme was expressed, and purified in Escherichia coli. Recombinant Gtb PAP2L2s from the membrane fractions were solublized with 0.3% (v/v) Triton X-100 and purified by Ni(2+) affinity chromatography. The purified enzyme showed broad substrate specificity to phosphatidic acid, diacylglycerol pyrophosphate, and lysophosphatidic, but preferred phosphatidic acid and diacylglycerol pyrophosphate in vitro. Gtb PAP2L2 is a thermal stable enzyme with a half-life of 30 min at 60 degrees C. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Zn(2+), whereas it was independent of the Mg(2+) ion, and insensitive to N-ethylmaleimide. The purified recombinant Gtb PAP2L2 was catalytically active and highly stable, making it ideal as a candidate on which to base further PAP2 structure/function studies.

Lipid phosphate phosphatases from Saccharomyces cerevisiae.

DPP1-encoded and LPP1-encoded lipid phosphate phosphatases are integral membrane proteins in the yeast Saccharomyces cerevisiae. They catalyze the Mg(2+)-independent dephosphorylation of bioactive lipid phosphate molecules such as diacylglycerol pyrophosphate and phosphatidate. These enzymes possess a three-domain lipid phosphatase motif that is localized to the hydrophilic surface of the membrane. The lipid phosphate phosphatase activities of DPP1-encoded and LPP1-encoded enzymes are measured by following the release of water-soluble radioactive inorganic phosphate from chloroform-soluble radioactive lipid phosphate substrate following a chloroform/methanol/water phase partition. The DPP1-encoded enzyme, commonly referred to as diacylglycerol pyrophosphate phosphatase, is purified from wild-type S. cerevisiae membranes by detergent solubilization with Triton X-100 followed by chromatography with DEAE-cellulose (DE53), Affi-Gel blue, hydroxylapatite, and Mono Q. The purification scheme yields an essentially homogeneous enzyme preparation that is stable for several years upon storage at -80 degrees . The properties of the DPP1-encoded and LPP1-encoded lipid phosphate phosphatase enzymes are summarized.

Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects.

A new procedure for the partial purification of Mg2+-dependent, N-ethylmaleimide-sensitive phosphatidate phosphohydrolase (Mg2+-PAP; EC 3.1.3.4) from rat liver cytosol is described, using protein precipitation with MgCl2, gel filtration on Sephacryl S-400, chromatography on DEAE-cellulose and affinity chromatography on calmodulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the beta-isoform of the 90-kDa heat shock protein, and Mg2+-PAP could be detected.

Molecular cloning and expression regulation of PRG-3, a new member of the plasticity-related gene family.

Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation.

Characterization and partial purification of CD34+ progenitor cell ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its hydrolysis product, diacylglycerol, play potentially vital roles as extracellular messengers in numerous cellular systems and may play a key role in regulating hematopoiesis. In this study, we describe an ecto-phosphatidic acid phosphohydrolase that potentially regulates cellular responses to phosphatidic acid on bone marrow derived human hematopoietic progenitors. We partially purified hematopoietic progenitor ecto-PAPase using a novel in-gel phosphatase assay and then characterized the enzyme on phenotypically defined subpopulations of hematopoietic CD34+ progenitors isolated by flow cytometry. The most pronounced PAPase activity was confined to uncommitted CD34+/CD38+ hematopoietic progenitors, which lacked the expression of other lineage-associated antigens. We conclude that hematopoietic progenitor cells at various stages of maturation possess a potent ecto-PAPase, an enzyme well positioned to regulate progenitor cell growth and differentiation induced by phosphatidic acid and related lipids.

Phosphatidate phosphatase--a key enzyme in glycerolipid biosynthesis. Studies on the yeast enzyme.

Phosphatidate phosphatase is an important enzyme in glycerolipid biosynthesis, but difficult to purify. A purified preparation of N-ethylmaleimide-sensitive phosphatidate phosphatase from the yeast Saccharomyces cerevisiae was obtained by a five-step protocol, using chromatography on DE-53/DEAE FF, Affi-Gel Blue, hydroxyapatite, Mono-Q, and Superdex 200. A protease-deficient yeast strain gave preparations similar to those of the wild-type strain. In exclusion chromatography, the enzyme activity of all preparations eluted at approximately the same position as albumin. However, the behavior on SDS/PAGE differed considerably among preparations, suggesting a multimeric subunit structure or degradation during purification. A 35-kDa and a 40-kDa protein band which coincided with activity were found in all preparations. Glycerol in the buffers could be excluded without rapid loss of enzyme activity, and Tris could be substituted for ammonium bicarbonate, while at least 0.6% sodium cholate in the buffers was essential.

Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli.

Phosphatidate phosphatase plays a major role in the synthesis of phospholipids and triacylglycerols in the yeast Saccharomyces cerevisiae. Membrane- and cytosolic-associated forms of the enzyme have been isolated and characterized. These enzymes are Mg2+-dependent and N-ethylmaleimide-sensitive. The expression of a membrane-associated form of phosphatidate phosphatase is regulated by growth phase and inositol supplementation, whereas enzyme activity is regulated by lipids, nucleotides, and by phosphorylation. Phosphatidate phosphatase is coordinately regulated with other phospholipid biosynthetic enzymes including phosphatidylserine synthase. Diacylglycerol pyrophosphate phosphatase is a novel enzyme of phospholipid metabolism which is present in S. cerevisiae, Escherichia coli, and mammalian cells. This enzyme possesses a phosphatidate phosphatase activity which is Mg2+-independent and N-ethylmaleimide-insensitive and is distinct from the Mg2+-dependent and N-ethylmaleimide-sensitive form of phosphatidate phosphatase. Genes encoding for diacylglycerol pyrophosphate phosphatase have been isolated from S. cerevisiae and E. coli. The deduced protein sequences of these genes show homology to the sequence of the mouse PAP2 (Mg2+-independent and N-ethylmaleimide-insensitive phosphatidate phosphatase) protein, especially in a novel phosphatase sequence motif. Rat liver PAP2 displays diacylglycerol pyrophosphate phosphatase activity.

Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 degrees C and pH7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1. 4x10(-3) M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin-Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

Mammalian Mg2+-independent phosphatidate phosphatase (PAP2) displays diacylglycerol pyrophosphate phosphatase activity.

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

Purification of a phosphatase which hydrolyzes phosphatidic acid, a key intermediate in glucolipid synthesis in Acholeplasma laidlawii A membranes.

A phosphatidic acid phosphatase (PAP; EC 3.1.3.4.), dephosphorylating phosphatidic acid (PA) to diacylglycerol (DAG), was identified and purified from the plasma membrane of Acholeplasma laidlawii A. After four purification steps, including membrane preparation, Tween 20 solubilization, preparative gel electrophoresis and electro-elution, PAP was purified about 400 times to near homogeneity. The molecular weight of PAP was according to SDS-polyacrylamide gel electrophoresis approximately 25 kDa and the enzyme was a stable and integral membrane protein. It is proposed to catalyze the first enzymatic step in the important glucolipid pathway of A. laidlawii. No essential cofactors or activator lipids were found. However, some divalent cations and phosphate analogues were potent inhibitors. Beside the in vivo substrate (PA), PAP was found to dephosphorylate p-nitrophenylphosphate. This less stringent specificity makes alternative in vivo functions for PAP plausible, the importance which is discussed.

Identification of phosphatidate phosphohydrolase purified from rat liver membranes on SDS-polyacrylamide gel electrophoresis.

Phosphatidate phosphohydrolase (PAP; EC 3.1.3.4) insensitive to N-ethylmaleimide was partially purified from rat liver membranes by a combination of chromatographic methods, immunoabsorption and glycerol gradient centrifugation. The specific activity was increased more than 600-fold over that of the membrane extract. Enzyme antibodies precipitating more than 80% of PAP were obtained and used for the identification of PAP protein on SDS-polyacrylamide gels employing the immunodetection method of Muilerman et al. [(1982) Anal. Biochem. 120, 46-51]. By this approach PAP was localized as a 31 kDa polypeptide.

Purification and characterization of novel plasma membrane phosphatidate phosphohydrolase from rat liver.

An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membranes of rat liver. The specific activity of an anionic form of the enzyme (53 kDa, pI < 4) was increased 2700-fold. A cationic form of enzyme (51 kDa, pI = 9) was purified to homogeneity, but the -fold purification was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneous protein confirmed the identity of the cationic protein as the phosphohydrolase and were used to identify the anionic enzyme. Both forms are integral membrane glycoproteins that were converted to 28-kDa proteins upon treatment with N-glycanase F. Treatment of the anionic form with neuraminidase allowed it to be purified in the same manner as the cationic enzyme and yielded an immunoreactive protein with a molecular mass identical to the cationic protein. Thus, the two ionic forms most likely represent different sialated states of protein. An immunoreactive 51-53-kDa protein was detected in rat liver, heart, kidney, skeletal muscle, testis, and brain. Little immunoreactive 51-53-kDa protein was detected in rat thymus, spleen, adipose, or lung tissue. This work provides the tools for determining the regulation and function of the phosphatidate phosphohydrolase in signal transduction and cell activation.

Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver.

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.