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The Plant Journal : For Cell and... Aug 2004Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response...
Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA-affinity chromatography. Several PA-binding proteins were present in the soluble fraction of tomato and Arabidopsis cells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14-3-3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA-binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo-osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA-affinity chromatography/mass spectrometry is an effective way to isolate and identify PA-binding proteins from plants.
Topics: Arabidopsis; Cells, Cultured; Chromatography, Affinity; Chromatography, Ion Exchange; Solanum lycopersicum; Mass Spectrometry; Phosphatidic Acids; Phosphoenolpyruvate Carboxylase; Plant Proteins; Protein Binding; Signal Transduction
PubMed: 15272872
DOI: 10.1111/j.1365-313X.2004.02152.x -
Traffic (Copenhagen, Denmark) Mar 2003The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown....
The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37 degrees C, pH 7.2, 0.5 mM free Mg2+), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca2+ (pH 6.6-5.9, 0.3 mM Ca2+). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cation-lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.
Topics: Calcium Chloride; Cell Division; Cell Size; Freeze Fracturing; Lysophospholipids; Magnesium Chloride; Magnetic Resonance Spectroscopy; Membrane Lipids; Models, Biological; Models, Molecular; Phosphatidic Acids; Phosphatidylcholines
PubMed: 12656989
DOI: 10.1034/j.1600-0854.2003.00086.x -
The Journal of Biological Chemistry Aug 1981The fatty acid composition of phosphatidylinositol (PI), phosphatidic acid (PA), and the free fatty acid pool of human platelets was studied as a function of time...
The fatty acid composition of phosphatidylinositol (PI), phosphatidic acid (PA), and the free fatty acid pool of human platelets was studied as a function of time following thrombin stimulation. Upon addition of thrombin, the total amount of fatty acids in PI decreased sharply, then rose toward basal levels, while that of PA showed an inverse pattern. However, the percentage distribution of fatty acids in stimulated as well as unstimulated PI and those in stimulated PA remained relatively constant: stearic and arachidonic acids accounted for 90 and 80% of the total in PI and PA, respectively. These data suggest that in stimulated human platelets PI and PA are interconverted via the "PI cycle." The time course of changes in PI and PA may suggest that the levels of arachidonyl-stearyl PA are involved in regulation of the resynthesis of arachidonyl-steryl PI. In contrast to PI and PA, the free fatty acid pool showed sharp increases in the five major platelet fatty acids: arachidonate, stearate, palmitate, oleate, as well as linoleate. These data suggest that most of the fatty acids liberated upon platelet stimulation are not derived via PI metabolism but by other mechanisms.
Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelets; Fatty Acids, Nonesterified; Humans; Kinetics; Phosphatidic Acids; Phosphatidylinositols; Stearic Acids; Thrombin
PubMed: 6790536
DOI: No ID Found -
Nature Chemical Biology Jan 2021TRAAK is an ion channel from the two-pore domain potassium (K) channel family with roles in maintaining the resting membrane potential and fast action potential...
TRAAK is an ion channel from the two-pore domain potassium (K) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K4.1 toward lipids remains poorly understood. Here we show the two isoforms of K4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K4.1. Liposome potassium flux assays with K4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K4.1.
Topics: Adenosine; Cations, Monovalent; Cloning, Molecular; Gene Expression; Genetic Vectors; Glycerophospholipids; Humans; Ion Channel Gating; Ion Transport; Kinetics; Liposomes; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Pichia; Potassium; Potassium Channels; Protein Binding; Protein Isoforms; Recombinant Proteins
PubMed: 32989299
DOI: 10.1038/s41589-020-00659-5 -
Molecular Pain May 2011Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin...
BACKGROUND
Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain.
RESULTS
(1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treatments with 2ccPA significantly attenuated thermal hyperalgesia and mechanical allodynia following partial ligation of the sciatic nerve. (5) In rats, repeated post-treatments with 2ccPA also significantly attenuated thermal hyperalgesia and mechanical allodynia following chronic sciatic nerve constriction.
CONCLUSIONS
Our results suggest that cPA and its stable analog 2ccPA inhibit chronic and acute inflammation-induced C-fiber stimulation, and that the central effects of 2ccPA following repeated treatments attenuate neuropathic pain.
Topics: Acute Disease; Anesthesia; Animals; Behavior, Animal; Chronic Disease; Cyclic P-Oxides; Disease Models, Animal; Electric Stimulation; Formaldehyde; Hyperalgesia; In Vitro Techniques; Injections, Intravenous; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Nociceptors; Pain; Phosphatidic Acids; Rats; Rats, Wistar; Reflex; Sympathetic Nervous System; Temperature
PubMed: 21569544
DOI: 10.1186/1744-8069-7-33 -
Biological & Pharmaceutical Bulletin 2021The aim of this study was to examine the effects of carba cyclic phosphatidic acid (ccPA) on cornified envelope (CE) formation and keratinocyte differentiation....
The aim of this study was to examine the effects of carba cyclic phosphatidic acid (ccPA) on cornified envelope (CE) formation and keratinocyte differentiation. ccPA-treated keratinocytes showed higher mRNA and protein levels of differentiation markers and CE components than untreated cells. These results suggest that ccPA could serve as therapeutic targets for treating skin barrier dysfunction because of their roles in upregulating genes and proteins associated with CE formation and keratinocyte differentiation.
Topics: Cell Differentiation; Cell Line; Cornified Envelope Proline-Rich Proteins; Gene Expression; Humans; Keratinocytes; Phosphatidic Acids
PubMed: 33642555
DOI: 10.1248/bpb.b20-00572 -
Developmental Cell Sep 2021The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid...
The cell nucleus is surrounded by a double membrane. The lipid packing and viscosity of membranes is critical for their function and is tightly controlled by lipid saturation. Circuits regulating the lipid saturation of the outer nuclear membrane (ONM) and contiguous endoplasmic reticulum (ER) are known. However, how lipid saturation is controlled in the inner nuclear membrane (INM) has remained enigmatic. Using INM biosensors and targeted genetic manipulations, we show that increased lipid unsaturation causes a reprogramming of lipid storage metabolism across the nuclear envelope (NE). Cells induce lipid droplet (LD) formation specifically from the distant ONM/ER, whereas LD formation at the INM is suppressed. In doing so, unsaturated fatty acids are shifted away from the INM. We identify the transcription circuits that topologically reprogram LD synthesis and identify seipin and phosphatidic acid as critical effectors. Our study suggests a detoxification mechanism protecting the INM from excess lipid unsaturation.
Topics: Cell Nucleus; Endoplasmic Reticulum; Fats, Unsaturated; GTP-Binding Protein gamma Subunits; Lipid Droplets; Lipid Metabolism; Membrane Proteins; Nuclear Envelope; Phosphatidic Acids; Yeasts
PubMed: 34407429
DOI: 10.1016/j.devcel.2021.07.018 -
Journal of Dairy Science Dec 1978Phosphatidic acid could not be demonstrated when milk was assayed for phospholipase D activity even when lecithin was added. The concentration of "free" choline in milk...
Phosphatidic acid could not be demonstrated when milk was assayed for phospholipase D activity even when lecithin was added. The concentration of "free" choline in milk remained the same before and after addition of lecithin. However, phosphatidic acid and an increased concentration of "free" choline were demonstrated when cabbage phospholipase D was added to raw milk. The concentration of phosphatidic acid and "free" choline in milk increased appreciably when both lecithin and cabbage phospholipase D were added to raw milk. Phospholipase D activity in milk was assayed at 23.5 C, pH 5.6, in the presence of 10% vol/vol of diethyl ether and .015M calcium chloride. "Free" choline was determined by adjusting the acid whey from milk to pH 11 and adding a freshly prepared 3% methanolic solution of ammonium reineckate. The precipitated choline reineckate was washed, dissolved in acetone, and its absorbance measured at 520 nm. The absence or presence of phosphatidic acid was shown by thin-layer chromatography. These results refute a previous report which proposed that both phosphatidic acid and phospholipase D were in milk.
Topics: Animals; Cattle; Milk; Phosphatidic Acids; Phospholipases
PubMed: 570572
DOI: 10.3168/jds.S0022-0302(78)83788-6 -
Proceedings of the National Academy of... Sep 1994Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular...
Recent experiments in several laboratories have provided evidence that phosphatidic acid functions in cell signaling. However, the mechanisms that regulate cellular phosphatidic acid levels remain obscure. Here we describe a soluble phospholipase A1 from bovine testis that preferentially hydrolyzes phosphatidic acid when assayed in Triton X-100 micelles. Moreover, the enzyme hydrolyzes phosphatidic acid molecular species containing two unsaturated fatty acids in preference to those containing a combination of saturated and unsaturated fatty acyl groups. Under certain conditions, the enzyme also displays lysophospholipase activity toward lysophosphatidic acid. The phospholipase A1 is not likely to be a lysosomal enzyme because its optimum pH is 7.5-8.5. Furthermore, it is probably not a general lipid metabolic enzyme because high levels of activity are found in mature testis and brain but no measurable activity is seen in liver, spleen, or heart. The fact that the activity of the phospholipase A1 in mature bovine testis is > 10-fold higher than that in newborn calf testis raises the possibility that the enzyme may play a regulatory role in spermatogenesis or sperm function.
Topics: Animals; Brain; Cattle; Chromatography, Ion Exchange; Cytosol; Kinetics; Male; Phosphatidic Acids; Phospholipases A; Phospholipases A1; Substrate Specificity; Testis
PubMed: 7937808
DOI: 10.1073/pnas.91.20.9574 -
The Journal of Biological Chemistry Nov 2015There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late,...
There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.
Topics: Animals; Calcium; Exocytosis; Membrane Fusion; Oocytes; Phosphatidic Acids; Phospholipase D; Strongylocentrotus purpuratus
PubMed: 26433011
DOI: 10.1074/jbc.M115.681429