Therapeutic potential of lipin inhibitors for the treatment of cancer.

Lipins are phosphatidic acid phosphatases (PAP) that catalyze the conversion of phosphatidic acid (PA) to diacylglycerol (DAG). Three lipin isoforms have been identified: lipin-1, -2 and -3. In addition to their PAP activity, lipin-1 and -2 act as transcriptional coactivators and corepressors. Lipins have been intensely studied for their role in regulation of lipid metabolism and adipogenesis; however, lipins are hypothesized to mediate several pathologies, such as those involving metabolic diseases, neuropathy and even cognitive impairment. Recently, an emerging role for lipins have been proposed in cancer. The study of lipins in cancer has been hampered by lack of inhibitors that have selectivity for lipins, that differentiate between lipin family members, or that are suitable for in vivo studies. Such inhibitors have the potential to be extremely useful as both molecular tools and therapeutics. This review describes the expression and function of lipins in various tissues and their roles in several diseases, but with an emphasis on their possible role in cancer. The mechanisms by which lipins mediate cancer cell growth are discussed and the potential usefulness of selective lipin inhibitors is hypothesized. Finally, recent studies reporting the crystallization of lipin-1 are discussed to facilitate rational design of novel lipin inhibitors.

Plant lipid phosphate phosphatases: current advances and future outlooks.

Lipids are widely distributed in various tissues of an organism, mainly in plant storage organs (e.g., fruits, seeds, etc.). Lipids are vital biological substances that are involved in: signal transduction, membrane biogenesis, energy storage, and the formation of transmembrane fat-soluble substances. Some lipids and related lipid derivatives could be changed in their: content, location, or physiological activity by the external environment, such as biotic or abiotic stresses. Lipid phosphate phosphatases (LPPs) play important roles in regulating intermediary lipid metabolism and cellular signal response. LPPs can dephosphorylate lipid phosphates containing phosphate monolipid bonds such as: phosphatidic acid, lysophosphatidic acid (LPA), and diacylglycerol pyrophosphate, etc. These processes can change the contents of some important lipid signal mediation such as diacylglycerol and LPA, affecting lipid signal transmission. Here, we summarize the research progress of LPPs in plants, emphasizing the structural and biochemical characteristics of LPPs and their role in spatio-temporal regulation. In the future, more in-depth studies are required to boost our understanding of the key role of plant LPPs and lipid metabolism in: signal regulation, stress tolerance pathway, and plant growth and development.

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities.

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP-HAD recombinant proteins in Escherichia coli.

The middle lipin domain adopts a membrane-binding dimeric protein fold.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

Lipid Phosphate Phosphatases and Cancer.

Lipid phosphate phosphatases (LPPs) are a group of three enzymes (LPP1-3) that belong to a phospholipid phosphatase (PLPP) family. The LPPs dephosphorylate a wide spectrum of bioactive lipid phosphates, among which lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are two important extracellular signaling molecules. The LPPs are integral membrane proteins, which are localized on plasma membranes and intracellular membranes, including the endoplasmic reticulum and Golgi network. LPPs regulate signaling transduction in cancer cells and demonstrate different effects in cancer progression through the breakdown of extracellular LPA and S1P and other intracellular substrates. This review is intended to summarize an up-to-date understanding about the functions of LPPs in cancers.

Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein.

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.

Casein kinase II-mediated phosphorylation of lipin 1ß phosphatidate phosphatase at Ser-285 and Ser-287 regulates its interaction with 14-3-3ß protein.

The mammalian lipin 1 phosphatidate phosphatase is a key regulatory enzyme in lipid metabolism. By catalyzing phosphatidate dephosphorylation, which produces diacylglycerol, the enzyme plays a major role in the synthesis of triacylglycerol and membrane phospholipids. The importance of lipin 1 to lipid metabolism is exemplified by cellular defects and lipid-based diseases associated with its loss or overexpression. Phosphorylation of lipin 1 governs whether it is associated with the cytoplasm apart from its substrate or with the endoplasmic reticulum membrane where its enzyme reaction occurs. Lipin 1ß is phosphorylated on multiple sites, but less than 10% of them are ascribed to a specific protein kinase. Here, we demonstrate that lipin 1ß is a bona fide substrate for casein kinase II (CKII), a protein kinase that is essential to viability and cell cycle progression. Phosphoamino acid analysis and phosphopeptide mapping revealed that lipin 1ß is phosphorylated by CKII on multiple serine and threonine residues, with the former being major sites. Mutational analysis of lipin 1ß and its peptides indicated that Ser-285 and Ser-287 are both phosphorylated by CKII. Substitutions of Ser-285 and Ser-287 with nonphosphorylatable alanine attenuated the interaction of lipin 1ß with 14-3-3ß protein, a regulatory hub that facilitates the cytoplasmic localization of phosphorylated lipin 1. These findings advance our understanding of how phosphorylation of lipin 1ß phosphatidate phosphatase regulates its interaction with 14-3-3ß protein and intracellular localization and uncover a mechanism by which CKII regulates cellular physiology.

Molecular insights into substrate binding mechanism of undecaprenyl pyrophosphate with membrane integrated phosphatidyl glycerophosphate phosphatase B (PgpB) using molecular dynamics simulation approach.

Undecaprenyl phosphate (C55-P) acts as carrier lipid in the synthesis of peptidoglycan, which is de novo synthesized from dephosphorylation of undecaprenyl pyrophosphate (C55-PP). The phosphatidylglycerol phosphate phosphatase B (PgpB) catalyzes the dephosphorylation of C55-PP and forms C55-P. As no structural study has been made regarding the binding of C55-PP to PgpB, in the current study, in silico molecular docking, followed by 150 ns molecular dynamics simulation of the putative binding complex in membrane/solvent environment has been performed to understand conformational dynamics. Results are compared with simulated apo form and PE inhibitor-bound form. Analysis of correlated residual fluctuation network in apo form, C55-PP bound and PE inhibitor-bound form suggests that difference in dynamic coupling between TM domain and α2 and α3 helix of periplasmic domain provides ligand binding to facilitate catalysis or to show inhibitory activity. Distance distribution in catalytic residual pair, H207-R104; H207-R201 and H207-D211 which stabilizes phosphate-enzyme intermediate shows a narrow peak in 2.4-3.6 Å in substrate-bound compared to apo form. Binding interactions and binding free energy analyses complement the partial inhibition of PE where PE has less binding free energy compared to the C55-PP substrate as well as the difference in binding interaction with catalytic pocket. Thus, the present study provides how substrate binding couples the movement in TM domain and periplasmic domain which might help in the understanding of active site communication in PgpB. C55-PP phosphatase interactions with a catalytic pocket of PgpB provide new insight for designing drugs against bacterial infection.

Fat-regulating phosphatidic acid phosphatase: a review of its roles and regulation in lipid homeostasis.

Phosphatidic acid (PA) phosphatase is an evolutionarily conserved enzyme that plays a major role in lipid homeostasis by controlling the cellular levels of its substrate, PA, and its product, diacylglycerol. These lipids are essential intermediates for the synthesis of triacylglycerol and membrane phospholipids; they also function in lipid signaling, vesicular trafficking, lipid droplet formation, and phospholipid synthesis gene expression. The importance of PA phosphatase to lipid homeostasis and cell physiology is exemplified in yeast, mice, and humans by a host of cellular defects and lipid-based diseases associated with loss or overexpression of the enzyme activity. In this review, we focus on the mode of action and regulation of PA phosphatase in the yeast Saccharomyces cerevisiae The enzyme Pah1 translocates from the cytosol to the nuclear/endoplasmic reticulum membrane through phosphorylation and dephosphorylation. Pah1 phosphorylation is mediated in the cytosol by multiple protein kinases, whereas dephosphorylation is catalyzed on the membrane surface by an integral membrane protein phosphatase. Posttranslational modifications of Pah1 also affect its catalytic activity and susceptibility to degradation by the proteasome. Additional mechanistic understanding of Pah1 regulation should be instrumental for the identification of small-molecule inhibitors or activators that can fine-tune PA phosphatase function and thereby restore lipid homeostasis.

Lipid phosphate phosphatase 3 in vascular pathophysiology.

LPP3 is an integral membrane protein belonging to a family of enzymes (LPPs) that display broad substrate specificity and catalyse dephosphorylation of several lipid substrates, including lysophosphatidic acid and sphingosine-1-phosphate. In mammals, the LPP family consists of three enzymes named LPP1, LPP2 and LPP3, which are encoded by three independent genes, PLPP1, PLPP2 and PLPP3, respectively (formerly known as PPAP2A, PPAP2C, PPAP2B). These three enzymes, in vitro, do not seem to differ for catalytic activities and substrate preferences. However, in vivo targeted inactivation of the individual genes has indicated that the enzymes do not have overlapping functions and that LPP3, specifically, plays a crucial role in vascular development. In 2011, two genome-wide association studies have identified PLPP3 as a novel locus associated with coronary artery disease susceptibility. Shortly after these reports, tissue specific inactivation of PLPP3 in mice highlighted a specific role for LPP3 in vascular pathophysiology and, more recently, in atherosclerosis development. This review is aimed at providing an updated overview on the function of LPP3 in embryonic cardiovascular development and on the experimental and clinical evidences relating this enzyme to vascular cell functions and cardiovascular disease.

Undecaprenyl phosphate metabolism in Gram-negative and Gram-positive bacteria.

Undecaprenyl phosphate (UP) is essential for the biosynthesis of bacterial extracellular polysaccharides. UP is produced by the dephosphorylation of undecaprenyl diphosphate (UPP) via de novo synthetic and recycling pathways. Gram-positive bacteria contain remarkable amounts of undecaprenol (UOH), which is phosphorylated to UP, although UOH has not been found in Gram-negative bacteria. Here, current knowledge about UPP phosphatase and UOH kinase is reviewed. Dephosphorylation of UPP is catalyzed by a BacA homologue and a type-2 phosphatidic acid phosphatase (PAP2) homologue. The presence of one of these UPP phosphatases is essential for bacterial growth. The catalytic center of both types of enzyme is located outside the cytoplasmic membrane. In Gram-positive bacteria, an enzyme homologous to DgkA, which is the diacylglycerol kinase of Escherichia coli, catalyzes UOH phosphorylation. The possible role of UOH and the significance of systematic construction of Staphylococcus aureus mutants to determine UP metabolism are discussed.

The phosphatidic acid-binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3.

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

A conserved tryptophan within the WRDPLVDID domain of yeast Pah1 phosphatidate phosphatase is required for its in vivo function in lipid metabolism.

PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase-like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions (e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions.

Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis.

The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.

Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

Role of lipid phosphate phosphatase 3 in human aortic endothelial cell function.

AIMS: Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase ß (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. METHODS AND RESULTS: To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. CONCLUSIONS: We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3.

Structural Insight into Substrate Selection and Catalysis of Lipid Phosphate Phosphatase PgpB in the Cell Membrane.

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro Here we report the crystal structure of the lipid-bound form of PgpB. The structure shows that a PE molecule is stabilized in a membrane-embedded tunnel formed by TM3 and the "PSGH" fingerprint peptide near the catalytic site, providing structural insight into PgpB substrate binding mechanism. Noteworthy, in silico docking of varied lipid phosphates exhibited similar substrate binding modes to that of PE, and the residues in the lipid tunnel appear to be important for PgpB catalysis. The catalytic triad in the active site is essential for dephosphorylating substrates lysophosphatidic acid, phosphatidic acid, or sphingosine-1-phosphate but surprisingly not for the native substrate PGP. Remarkably, residue His-207 alone is sufficient to hydrolyze PGP, indicating a specific catalytic mechanism for PgpB in PG biosynthesis. We also identified two novel sensor residues, Lys-93 and Lys-97, on TM3. Our data show that Lys-97 is essential for the recognition of lyso-form substrates. Modification at the Lys-93 position may alter substrate specificity of lipid phosphate phosphatase proteins in prokaryotes versus eukaryotes. These studies reveal new mechanisms of lipid substrate selection and catalysis by PgpB and suggest that the enzyme rests in a PE-stabilized state in the bilayer.

Cloning and characterization of three Eimeria tenella lipid phosphate phosphatases.

Although lipid phosphate phosphatases (LPPs) play an important role in cellular signaling in addition to lipid biosynthesis, little is thus far known about parasite LPPs. In this study, we characterized three Eimeria tenella cDNA clones encoding LPP named EtLPP1, EtLPP2 and EtLPP3. Key structural features previously described in LPPs, including the three conserved domains proposed as catalytic sites, a single conserved N-glycosylation site, and putative transmembrane domains were discovered in the three resulting EtLPP amino acid sequences. Expression of His6-tagged EtLPP1, -2, and -3 in HEK293 cells produced immunoreactive proteins with variable molecular sizes, suggesting the presence of multiple forms of each of the three EtLPPs. The two faster-migrating protein bands below each of the three EtLPP proteins were found to be very similar to the porcine 35-kDa LPP enzyme in their molecular size and the extent of their N-glycosylation, suggesting that the three EtLPPs are partially N-glycosylated. Kinetic analyses of the activity of the three enzymes against PA, LPA, C1P and S1P showed that Km values for each of the substrates were (in µM) 284, 46, 28, and 22 for EtLPP1; 369, 179, 237, and 52 for EtLPP2; and 355, 83, and 260 for EtLPP3. However, EtLPP3 showed negligible activity on S1P. These results confirmed that the three EtLPPs have broad substrate specificity. The results also indicated that despite structural similarities, the three EtLPPs may play distinct functions through their different models of substrate preference. Furthermore, particularly high expression levels of the three EtLPP genes were detected in the sporozoite stage of the E. tenella life cycle (p<0.001), suggesting that their encoded proteins might play an important biological function in the sporozoite stage.

Phosphorylation regulates the ubiquitin-independent degradation of yeast Pah1 phosphatidate phosphatase by the 20S proteasome.

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.

Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).

Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. The fruit has been widely used as vegetable and herbal medicine. Alpha-eleostearic acid is the major fatty acid in the seeds, but little is known about its biosynthesis. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and P(i). PAPs are typically categorized into two subfamilies: Mg(2+)-dependent soluble PAP and Mg(2+)-independent membrane-associated PAP. We report here the partial purification and characterization of an Mg(2+)-independent PAP activity from developing cotyledons of bitter melon. PAP protein was partially purified by successive centrifugation and UNOsphere Q and S columns from the soluble extract. PAP activity was optimized at pH 6.5 and 53-60 °C and unaffected by up to 0.3 mM MgCl2. The K(m) and Vmax values for dioleoyl-phosphatidic acid were 595.4 µM and 104.9 ηkat/mg of protein, respectively. PAP activity was inhibited by NaF, Na(3)VO(4), Triton X-100, FeSO4 and CuSO4, but stimulated by MnSO4, ZnSO4 and Co(NO3)2. In-gel activity assay and mass spectrometry showed that PAP activity was copurified with a number of other proteins. This study suggests that PAP protein is probably associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg(2+)-independent enzyme in plants.