Lysophospholipid acyltransferases and leukotriene biosynthesis: intersection of the Lands cycle and the arachidonate PI cycle

Considerable information is now available concerning the complexity of biochemical events that take place leading to the synthesis of leukotrienes (LTs), which are lipid mediators derived from arachidonic acid (AA). However, important questions remain concerning LT biosynthesis before a complete description of the function that leukotrienes play in both human health and disease can be made. With our expanding understanding that inflammation plays critical roles in multiple disease processes, leukotrienes continue to be recognized as important substances to consider in events taking place in pathophysiology. Leukotrienes are the result of several steps of enzymatic processing of the initial, chemically unstable leukotriene A4 (LTA4). LTA4 is the direct chemical precursor of leukotriene B4 (LTB4), a chemotactic lipid (1Haeggström J.Z. Wetterholm A. Enzymes and receptors in the leukotriene cascade.Cell. Mol. Life Sci. 2002; 59: 742-753Crossref PubMed Scopus (79) Google Scholar, 2Rådmark O. Werz O. Steinhilber D. Samuelsson B. 5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease.Biochim. Biophys. Acta. 2015; 1851: 331-339Crossref PubMed Scopus (329) Google Scholar), or leukotriene C4 (LTC4) and peptide cleavage products leukotriene D4 (LTD4) and leukotriene E4 (LTE4), which constitute the biological activity termed slow reacting substance of anaphylaxis (3Murphy R.C. Hammarström S. Samuelsson B. Leukotriene C: a slow reacting substance (SRS) from murine mastocytoma cells.Proc. Natl. Acad. Sci. USA. 1979; 76: 4275-4279Crossref PubMed Scopus (872) Google Scholar). Therefore, investigations into the basic biochemistry engaged in leukotriene biosynthesis, release of these specific lipids from the cell, and intracellular signaling events initiated from leukotriene receptor occupation continue to be important. Five-lipoxygenase (5-LO) is a nonheme iron dioxygenase, with a C-2 domain at the N terminus and an active site C terminus made up of α-helices, that stereospecifically attaches diatomic oxygen to the 5-carbon position of free AA. Specific details concerning structure of this enzyme and the nature of the histidine coordination of iron (4Gilbert N.C. Bartlett S.G. Waight M.T. Boeglin W.E. Brash A.R. Newcommer M.E. The structure of human 5-lipoxygenase.Science. 2011; 331: 217-219Crossref PubMed Scopus (318) Google Scholar), redox changes that occur during the lipoxygenase reaction (5Chasteen N.D. Grady J.K. Skorey K.I. Neden K.J. Riendeau D. Percival M.D. Characterization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous and ferric states.Biochemistry. 1993; 32: 9763-9771Crossref PubMed Scopus (49) Google Scholar), sites for protein phosphorylation (6Brock T. Regulating leukotriene synthesis: the role of 5-lipoxygenase.J. Chem. Biochem. 2005; : 1203-1211Google Scholar, 7Luo M. Jones S.M. Phare S.M. Coffey M.J. Peters-Golden M. Brock T.G. Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523.J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), as well as substrate binding (4Gilbert N.C. Bartlett S.G. Waight M.T. Boeglin W.E. Brash A.R. Newcommer M.E. The structure of human 5-lipoxygenase.Science. 2011; 331: 217-219Crossref PubMed Scopus (318) Google Scholar) are now known. There is limited expression of the gene that codes for 5-LO, which is somewhat restricted to inflammatory cells such as neutrophils, mast cells, eosinophils, macrophages and basophils. There is some evidence that 5-LO can be expressed in virus- and cancer-transformed cells (8Gentile D. Evolving role of leukotrienes in the pathogenesis of viral infections, including otitis media.Curr. Allergy Asthma Rep. 2006; 6: 316-320Crossref PubMed Scopus (3) Google Scholar, 9Wang D.R.N. Dubois R.N. Eicosanoids and cancer.Nat. Rev. Cancer. 2010; 10: 181-193Crossref PubMed Scopus (1295) Google Scholar). In the context of this review, it is important to consider that 5-LO is typically, but not exclusively, found in the cytosol of cells that express the 5-LO gene. For the enzymatic reaction to proceed, cytosolic 5-LO must be translocated to the perinuclear membrane. This translocation event is a result of the elevation of intracellular calcium ions also required to activate the enzyme 5-LO. Recent studies have suggested that the translocation process is mediated by a coactosin-like protein that binds 5-LO as well as F-actin and calcium ions critical for translocation and 5-LO activation (10Rakonjac M. Fischer L. Provost P. Werz O. Steinhilber D. Samuelsson B. Rådmark O. Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production.Proc. Natl. Acad. Sci. USA. 2006; 103: 13150-13155Crossref PubMed Scopus (83) Google Scholar). This elevation of intracellular calcium ion is the result of many different types of cell stimulation that initiate leukotriene biosynthesis. Once at the nuclear envelope, a second binding protein, termed five-lipoxygenase activating protein, is engaged and thought to act as a scaffolding protein (11Evans J.F. Ferguson A.D. Mosley R.T. Hutchinson J.H. What's all the FLAP about? 5-lipoxygenase-activating protein inhibitors for inflammatory diseases.Trends Pharmacol. Sci. 2008; 29: 72-78Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) as well as presenting AA to the catalytic site of 5-LO. In addition to elevated calcium ions, free AA must appear within the nuclear membrane bilayer as the required substrate for 5-LO. This elevation of free AA at the nuclear dual bilayer is a result of the action of phospholipase A2 that hydrolyzes esterified arachidonate at the sn-2 position of phospholipids. There are a large number of phospholipase A2s (12Astudillo A.M. Balboa M.A. Balsinde J. Selectivity of phospholipid hydrolysis by phospholipase A2 enzymes in activated cells leading to polyunsaturated fatty acid mobilization.Biochim. Biophys. Acta Mol. Cell. Biol. Lipids. 2018; PubMed Google Scholar) and likely multiple enzymes are engaged in the series of events that result from liberation of free AA. It is now recognized that a cytosolic phospholipase A2, cPLA2α, is critical for the biosynthesis of leukotrienes (13Leslie C.C. Cytosolic phospholipase A2: physiological function and role in disease.J. Lipid Res. 2015; 56: 1386-1402Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Cytosolic cPLA2α is also translocated to the nuclear envelope, a result of elevation of intracellular calcium during cell stimulation. A typical pathway summary for leukotriene biosynthesis is diagrammed in Fig. 1, but this is known to be a rather oversimplification of the complexity of events that actually take place leading to the activation of 5-LO, translocation and conversion of free AA into leukotriene A4 at the nuclear bilayer. In addition to the requirements for calcium ion elevation and free AA, 5-LO activity can be regulated by protein phosphorylation at Ser271 (MAPKAP kinase site), Ser663 (ERK2 site), and Ser523 (PKA site) (6Brock T. Regulating leukotriene synthesis: the role of 5-lipoxygenase.J. Chem. Biochem. 2005; : 1203-1211Google Scholar, 7Luo M. Jones S.M. Phare S.M. Coffey M.J. Peters-Golden M. Brock T.G. Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523.J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The latter site reduces activity of 5-LO in a cAMP dependent manner, likely by altering translocation of 5-LO to the nuclear membrane (14Flamand N. Surette M.E. Picard S. Bourgoin S. Borgeat P. Cyclic AMP-mediated inhibition of 5-lipoxygenase translocation and leukotriene biosynthesis in human neutrophils.Mol. Pharmacol. 2002; 62: 250-256Crossref PubMed Scopus (110) Google Scholar). Thus, agents that affect cAMP, such as adenosine (15Flamand N.S. Boudreault S. Picard M. Austin M.E. Surette H. Plante E. Krump M.J. Vallée C. Gilbert P. Naccache M. et al.Adenosine, a potent natural suppressor of arachidonic acid release and leukotriene biosynthesis in human neutrophils.Am. J. Respir. Crit. Care Med. 2000; 161: S88-S94Crossref PubMed Scopus (75) Google Scholar), steroids (16Riddick C.A. Ring W.L. Baker J.R. Hodulik C.R. Bigby T.D. Dexamethasone increases expression of 5-lipoxygenase and its activating protein in human monocytes and THP-1 cells.Eur. J. Biochem. 1997; 246: 112-118Crossref PubMed Scopus (95) Google Scholar), and β2 adrenergic agonists (17Fonteh A.N. Winkler J.D. Torphy T.J. Heravi J. Undem B.J. Chilton F.H. Influence of isoproterenol and phosphodiesterase inhibitors on platelet-activating factor biosynthesis in the human neutrophil.J. Immunol. 1993; 151: 339-350PubMed Google Scholar), can reduce production of leukotrienes, as has been known for some time (18Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl F.A. Jr F.A. Inhibition by prostaglandins of leukotriene B4 release from activated neutrophils.Proc. Natl. Acad. Sci. USA. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar). The production of specific leukotrienes, such as LTB4 or LTC4, is the result of enzymatic conversion of LTA4 (the direct 5-LO product) by leukotriene A4 hydrolase (19Wan M. Tang X. Stsiapanava A. Haeggström J.Z. Biosynthesis of leukotriene B4.Semin. Immunol. 2017; 33: 3-15Crossref PubMed Scopus (42) Google Scholar) and leukotriene C4 synthase (20Kanaoka Y. Boyce J.A. Cysteinyl leukotrienes and their receptors; emerging concepts.Allergy Asthma Immunol. Res. 2014; 6: 288-295Crossref PubMed Scopus (93) Google Scholar), respectively. LTC4 synthase also requires a cosubstrate, the tripeptide GSH. Once synthesized, these lipids leave the cytosol (LTB4) or nuclear membrane (LTC4), likely mediated by a transport carrier, and exit the cell, where they can bind to a specific G-protein membrane receptor to elicit a biological action. Peptidases are responsible for cleavage of the GSH tripeptide into LTD4 and LTE4 as the result of a specific γ-glutamyl transpeptidase (21Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.Z. Wan D.F. gamma-Glutamyl transpeptidase. What does the organization and expression of a multipromoter gene tell us about its functions?.Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar) and a unique dipeptidase (22Lee C.W. Lewis R.A. Corey E.J. Austen K.F. Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes.Immunology. 1983; 48: 27-35PubMed Google Scholar). Expression of these auxiliary enzymes is an important determinant of which specific leukotriene is made by an individual cell and, therefore, the biological activities that result from activation of the 5-LO pathway in a tissue. A rather unexpected mechanism by which leukotrienes appear in a tissue is that of movement of newly synthesized LTA4 from the activated cell that expressed 5-LO into a cell that contains one of the auxiliary enzymes, such as LTC4 synthase or LTA4 hydrolase (23Folco G. Murphy R.C. Eicosanoid transcellular biosynthesis: from cell-cell interactions to in vivo tissue responses.Pharmacol. Rev. 2006; 58: 375-388Crossref PubMed Scopus (187) Google Scholar). This is the process of transcellular biosynthesis. The curious feature of such events is that LTA4 is a conjugated triene epoxide and as such, quite chemically reactive. This epoxide has been suggested to have a chemical half-life under 1 s (24Fitzpatrick F.A. Morton D.R. Wynalda M.A. Albumin stabilizes leukotriene A4.J. Biol. Chem. 1982; 257: 4680-4683Abstract Full Text PDF PubMed Google Scholar). Considering that LTA4 is made at the nuclear envelope, it must cross not only the cytosol but also at least two plasma membranes to find the site within another cell where LTA4 hydrolase or LTC4 synthase reside. For this lipid to survive the aqueous environment, it must be protected, most likely by protein binding in a hydrophobic pocket (Fig. 1). Several candidate proteins have been proposed (25Dickinson Zimmer J. Bernlohr D.A. Murphy R.C. Fatty acid-binding proteins: stabilization of leukotriene A4 and competition with arachidonic acid.J. Lipid Res. 2004; 45: 2138-2144Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), with the most effective stabilizing protein being albumin (24Fitzpatrick F.A. Morton D.R. Wynalda M.A. Albumin stabilizes leukotriene A4.J. Biol. Chem. 1982; 257: 4680-4683Abstract Full Text PDF PubMed Google Scholar). Albumin is also shown to stabilize another very unstable AA-metabolite, i.e., thromboxane A2 (26Folco G. Granström E. Kindahl H. Albumin stabilizes thromboxane A2.FEBS Lett. 1977; 82: 321-324Crossref PubMed Scopus (48) Google Scholar). An interesting question that has emerged concerning transcellular biosynthesis is whether or not LTA4 is bound to a carrier protein in the extracellular media or, rather, whether cell-cell contact is required for a stabilizing protein to transfer LTA4 into a recipient cell. In experiments attempting to address this question, various antibodies to one potential carrier protein, complex S100 A8/A9 (27Rector C.L. Murphy R.C. Determination of leukotriene A4 stabilization by S100A8/A9 proteins using mass spectrometry.J. Lipid Res. 2009; 50: 2064-2071Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar), were added to suspensions of human neutrophils to trap any extracellular LTA4 bound to these proteins. The result of these experiments was unexpected in that, rather than increasing the amount of nonenzymatic transformation of LTA4 into 6-trans LTB4, the total synthesis of LTB4 increased almost 50-fold. Such a promotion of stimulated leukotriene biosynthesis had never been previously observed, requiring some understanding of the mechanism by which this occurred. After considerable effort, it was found that the antibody itself was not involved, but, rather, a preservative in the antibody preparation was responsible for this stimulation of leukotriene biosynthesis. This preservative was thimerosal, an organomercury reagent (ethyl (2-mercaptobenzoato- (2Rådmark O. Werz O. Steinhilber D. Samuelsson B. 5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease.Biochim. Biophys. Acta. 2015; 1851: 331-339Crossref PubMed Scopus (329) Google Scholar)-O,S) mercurate), used in the past to preserve vaccines. It was found that thimerosal substantially increased the level of free AA in stimulated macrophages and neutrophils, likely by inhibiting a lysophospholipid acyltransferase (LPCAT) responsible for the conversion of arachidonoyl CoA into an arachidonate containing phospholipid (28Zarini S. Gijon M.A. Folco G. Murphy R.C. Effect of arachidonic acid reacylation on leukotriene biosynthesis in human neutrophils stimulated with granulocyte-macrophage colony-stimulating factor and formyl-methionyl-leucyl-phenylalanine.J. Biol. Chem. 2006; 281: 10134-10142Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The unexpected nature of this serendipitous finding revealed the involvement of the reacylation pathway termed the Lands pathway (29Wang B. Tontonoz P. Phospholipid remodeling in physiology and disease.Annu. Rev. Physiol. 2018; PubMed Google Scholar) in leukotriene biosynthesis. Thimerosal had been previously suggested to inhibit lysophosphatidylcholine acyltransferase as well as transacylases (30Kröner E.E. Peskar B.A. Fischer H. Ferber E. Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases.J. Biol. Chem. 1981; 256: 3690-3697Abstract Full Text PDF PubMed Google Scholar, 31Förstermann U. Goppelt-Strübe M. Frölich J.C. Busse R. Inhibitors of acyl-coenzyme A:lysolecithin acyltransferase activate the production of endothelium-derived vascular relaxing factor.J. Pharmacol. Exp. Ther. 1986; 238: 352-359PubMed Google Scholar, 32Kaever V. Firla U. Resch K. Sulfhydryl reagents as model substances for eicosanoid research.Eicosanoids. 1988; 1: 49-57PubMed Google Scholar). A detailed understanding of exactly which LPCATs were present in the human neutrophils was at that time unknown. The Lands pathway involves remodeling of phospholipids synthesized by the normal Kennedy pathway, which generates saturated and monounsaturated fatty acyl groups on the various phospholipid backbones (Fig. 2). In order to incorporate polyunsaturated fatty acids derived from the diet into phospholipids, a phospholipase A2 cleaves the fatty acyl group from the sn-2 position of the de novo synthesized phospholipid forming a lysophospholipid for each of the choline-, inositol-, ethanolamine-, glycerol-, and serine-phospholipid classes, then arachidonoyl CoA is covalently bound to the lysophospholipid through the action of one or more LPCATs. There are numerous LPCATs (33Yamashita A. Hayashi Y. Nemoto-Sasaki Y. Ito M. Oka S. Tanikawa T. Waku K. Sugiura T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms.Prog. Lipid Res. 2014; 53: 18-81Crossref PubMed Scopus (164) Google Scholar), each with somewhat specific roles for the formation of specific phospholipid molecular species, including polyunsaturated fatty acyl containing phospholipids. The fatty acyl CoA ester is derived from the free fatty acid covalently linked as a thioester to CoA by a long chain-fatty acyl CoA synthase (ACSL) in a two-step mechanism (34Grevengoed T.J. Klett E.L. Coleman R.A. Acyl-CoA metabolism and partitioning.Annu. Rev. Nutr. 2014; 34: 1-30Crossref PubMed Scopus (228) Google Scholar). There are several families of these synthases known to be present in mammalian cells and responsible for the formation of the various long chain fatty acyl CoA esters. Although ACSLs appear to have considerable promiscuity, some experiments suggest that ACSL-4 is somewhat specific for formation of arachidonate CoA esters (35Tuohetahuntila M. Spee B. Kruitwagen H.S. Wubbolts R. Brouwers J.F. van de Lest C.H. Molenaar M.R. Houweling M. Helms J.B. Vaandrager A.B. Role of long-chain acyl-CoA synthetase 4 in formation of polyunsaturated lipid species in hepatic stellate cells.Biochim. Biophys. Acta. 2015; 1851: 220-230Crossref PubMed Scopus (26) Google Scholar). In addition to the synthases, there are also fatty acyl CoA hydrolases, which hydrolyze the CoA esters back to the free fatty acids (36Hunt M.C. Alexson S.E. The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism.Prog. Lipid Res. 2002; 41: 99-130Crossref PubMed Scopus (218) Google Scholar). The fatty acyl CoA thioesters are engaged in many lipid biosynthetic reactions including neutral lipid formation, such as cholesteryl ester, and triacylglycerol biosynthesis (37Coleman R.A. Mashek D.G. Mammalian triacylglycerol metabolism: synthesis, lipolysis, and signaling.Chem. Rev. 2011; 111: 6359-6386Crossref PubMed Scopus (172) Google Scholar). The fatty acyl CoA thioesters are also required for β-oxidation, either in the mitochondria or peroxisome (38Hunt M.C. Tillander V. Alexson S.E. Regulation of peroxisomal lipid metabolism: the role of acyl-CoA and coenzyme A metabolizing enzymes.Biochimie. 2014; 98: 45-55Crossref PubMed Scopus (61) Google Scholar). Polyunsaturated fatty acyl CoA esters are the required substrates of LPCATs along with a lysophospholipid in the synthesis of a polyunsaturated phospholipid molecular species. The LPCAT genes expressed in the human neutrophil are MBOAT1, MBOAT2, MBOAT5, and MBOAT7 (39Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Alternative designation of these proteins have been recently suggested as LPEAT1, LPCAT4, LPCAT3, and LPIAT, respectively (40Shindou H. Hishikawa D. Harayama T. Eto M. Shimizu T. Generation of membrane diversity by lysophospholipid acyltransferases.J. Biochem. 2013; 154: 21-28Crossref PubMed Scopus (93) Google Scholar). The substrate specificity of these enzymes was investigated using a novel substrate competition assay that involved incubation of a mutant yeast (Ale1 deficient) engineered to express each of these human MBOATs with six different fatty acyl CoA esters and eight different lysophospholipid substrates in a single in vitro assay. Because the mutant yeast (Ale1) lacked any lysophospholipid acyltransferase activity, this experiment revealed which substrates each human LPCAT preferred. The results were fascinating in that two enzymes were found to be rather specific for AA incorporation into phospholipids, LPCAT3/MBOAT5 and MBOAT7 (39Gijón M.A. Riekhof W.R. Zarini S. Murphy R.C. Voelker D.R. Lysophospholipid acyltransferases and arachidonate recycling in human neutrophils.J. Biol. Chem. 2008; 283: 30235-30245Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The question of whether or not an LPCAT can control leukotriene biosynthesis has been addressed using several different experimental approaches. As discussed above, the drug thimerosal had a profound effect on increasing leukotriene A4 biosynthesis in the human neutrophil. Lipidomic analysis revealed substantial changes in neutrophil phospholipid molecular species containing arachidonate after thimerosal exposure (unpublished observations). In an experiment designed to probe the effect of a chronic loss of one LPCAT specific for arachidonate remodeling, a lentivirus shRNA was used to knock down LPCAT3/MBOAT5 in RAW264.7 cells (41Martin S.A. Gijon M.A. Voelker D.R. Murphy R.C. Measurement of lysophospholipid acyltransferase activities using substrate competition.J. Lipid Res. 2014; 55: 782-791Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Following cell stimulation, phospholipid molecular species, free AA, and LTC4 analysis was carried out. Three different shRNA constructs were made that reduced the expression of LPCAT3/MBOAT5. Lipidomic analysis of the control and knockdown cells revealed substantial decrease in arachidonate-PE as well as arachidonate-PC as expected (supplemental Fig. S1). However, only one of the shRNA constructs (construct number 1) altered the production of LTC4 (supplemental Fig. S2) after stimulation with 3 mM ATP for 15 min. There was no change in the expression of ACSL, 5-LO, or cPLA2α in these LPCAT3/MBOAT5 knockdown RAW cells (41Martin S.A. Gijon M.A. Voelker D.R. Murphy R.C. Measurement of lysophospholipid acyltransferase activities using substrate competition.J. Lipid Res. 2014; 55: 782-791Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Overall, the results of these experiments were somewhat unexpected in that there did appear to be major change in PE and PC phospholipid remodeling, but there was not a significant change in production of LTC4. PI molecular species containing arachidonate, however, were not significantly altered in these knockdown cells. Based on a large number of mass spectrometric-based lipidomic studies, the major molecular species of PI observed in mammalian cells appears at m/z 885.5494, the negative ion corresponding to PI(38:4), which has been analyzed by tandem mass spectrometry and identified as PI(18:0/20:4) (42Zemski Berry K.A. Murphy R.C. Kosmider B. Mason R.J. Lipidomic characterization and localization of phospholipids in the human lung.J. Lipid Res. 2017; 58: 926-933Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). However, in almost all lipidomic studies, subcellular location of PI molecular species is not examined, but, rather, total cellular PI molecular species reported. Yet, considerable interest in PI has focused on the plasma membrane where receptor signaling events take place. Formation of second messengers are derived from PI-4,5P2 (PIP2) by the action of phospholipase C, which catalyzes the hydrolysis of this phospholipid into inositol trisphosphate (IP3) and diacylglycerol that then appear at the plasma membrane (Fig. 3) (43O'Donnell V.B. Rossjohn J. Wakelam M.J. Phospholipid signaling in innate immune cells.J. Clin. Invest. 2018; 128: 2670-2679Crossref PubMed Scopus (48) Google Scholar). These facts support the concept that another important arachidonate phospholipid cycle exists, one that involves the plasma membrane PI(18:0/20:4)-4,5P2. This phosphoinositol molecular species is formed from PI(18:0/20:4) made in the ER/trans Golgi by phosphatidylinositol synthase then transported to the plasma membrane, where phosphorylation occurs by two kinases, PI4 kinase and PI4P-5 kinase (44Kolay S. Basu U. Raghu P. Control of diverse subcellular processes by a single multi-functional lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2].Biochem. J. 2016; 473: 1681-1692Crossref PubMed Scopus (50) Google Scholar). After release of IP3, the diglyceride, DG (18:0/20:4), is transported back to the ER/trans Golgi (45Ueda Y. Ishitsuka R. Hullin-Matsuda F. Kobayashi T. Regulation of the transbilayer movement of diacylglycerol in the plasma membrane..Biochimie. 2014; Crossref PubMed Scopus (10) Google Scholar) and phosphorylated to form the phosphatidic acid molecular species that is subsequently converted to CDP-DG (18:0/ 20:4), thus completing the arachidonate PI cell cycle. The initial appearance of AA in this arachidonate PI cycle is undoubtedly a result of intersection of this pathway with the Lands pathway of phospholipid remodeling, residing also at the ER. MBOAT7 in the ER/trans Golgi specifically binds arachidonoyl-CoA ester to lyso PI(18:0) to form the abundant molecular species PI(18:0/20:4). Formation of lyso PI(18:0) would involve one or more phospholipase A2 species at the ER or even the nuclear membrane. Previous work has found that the subcellular location of PI molecular species is not uniform. The highest abundance of PI is found divided between the ER and the plasma membrane, and very small amounts at the nuclear membrane (supplemental Fig. S3) (46Andreyev A.Y. Fahy E. Guan Z. Kelly S. Li X. McDonald J.G. Milne S. Myers D. Park H. Ryan A. et al.Subcellular organelle lipidomics in TLR-4-activated macrophages.J. Lipid Res. 2010; 51: 2785-2797Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This very high level of PI in the plasma membrane is probably a consequence of the importance of the PI signaling cascade in cellular biochemistry. The importance of PI to the signaling cascade has also been suggested by the failure of MBOAT7 knockout mice to survive (47Lee H.C. Inoue T. Sasaki J. Kubo T. Matsuda S. Nakasaki Y. Hattori M. Tanaka F. Udagawa O. Kono N. et al.LPIAT1 regulates arachidonic acid content in phosphatidylinositol and is required for cortical lamination in mice.Mol. Biol. Cell. 2012; 23: 4689-4700Crossref PubMed Scopus (93) Google Scholar). It is widely appreciated that de novo phospholipid biosynthesis and the Lands cycle remodeling pathway are restricted to the intracellular ER location (29Wang B. Tontonoz P. Phospholipid remodeling in physiology and disease.Annu. Rev. Physiol. 2018; PubMed Google Scholar). Once the synthesis and remodeling of phospholipids into new molecular species has taken place, these phospholipids find their way to various organelles such as the plasma membrane and nuclear membranes suggested by the rather simplistic diagrams presented above (Figs. 2, 3). Yet, phospholipids and even fatty acids are poorly soluble in water and thus unlikely to diffuse by passive mechanisms, certainly not in a rapid fashion. Rather, it is becoming increasingly clear that protein-mediated events are involved to rapidly move lipids between various subcellular organelles. A host of proteins are known to be involved in this process of insoluble lipid movement within the cell. For example, fatty acids are transported by binding protein such as fatty acid translocases (FAT/CD36), fatty acid binding proteins, fatty acid transport proteins, as well as ubiquitous proteins such as intracellular albumin (48Duttaroy A.K. Transport of fatty acids across human placenta: a review.Prog. Lipid Res. 2009; 48: 52-61Crossref PubMed Scopus (221) Google Scholar). The mechanism of protein-mediated transfer of intact phospholipids has been suggested to be largely the result of physical membrane contact (49Hanada K. Lipid transfer proteins rectify inter-organelle flux and actively deliver lipids at membrane contact sites.J. Lipid Res. 2018; 59: 1341-1366Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The consequence of protein involvement in membrane-associated transfer of fatty acid and phospholipid molecular species is that each organelle has a unique phospholipid composition at any one time. Phospholipid molecular species and free fatty acids are not in chemical equilibrium within the cell, and the disequilibrium is maintained through protein binding and transfer, likely through direct organelle contact. The stimulation of leukotriene production in cells treated with thimerosal implies that a substantial concentration of free AA appears rapidly in the nuclear membrane, the site in which 5-LO forms LTA4. This excess free AA, as well as the lysophospholipids formed by the action of phospholipase A2, under normal circumstances would be immediately transported to the ER where there is enzymatic activity for remodeling by the Lands cycle (LPCATs and ACSL to form arachidonoyl CoA). However, when thimerosal is present to inhibit ER LPCATs, the level of free AA and arachidonoyl CoA increase in the ER, which slows transfer of nuclear membrane free AA. Thus, at the nuclear membrane, free AA increases dramatically. Interestingly, it had never been appreciated that 5-LO-mediated production of LTA4 could be mediated by the disappearance of free AA from the nuclear membrane and transfer to the ER for Lands cycle reesterification. Disruption of these lipid transport events and altered arachidonoyl CoA concentration at the ER permitted uncontrolled LTA4 formation due to the continued high abundance of free AA within the nuclear membrane. In terms of knockdown experiments or specific inhibition of one acyltransferase activity, the buildup of free AA in the ER would be unlikely because multiple enzymes are present there to use the excess arachidonoyl CoA as a substrate. Specifically, in the MBOAT5 knockdown experiments, the PI(18:0/20:4) synthesis and active MBOAT7 remain in place at the ER to use excess arachidonoyl CoA derived from the cPLA2 release of AA at the nuclear membrane. Any specific decrease in MBOAT7 activity at the ER would be expected to result in a reduction of LTA4 formation by a decrease in nuclear membrane PI(18:0/20:4) content, but certainly not an increase of LTA4 formation observed in the thimerosal experiments where multiple LPCATs were inhibited. We suggest that arachidonate-containing PI (PIP and PIP2) abundance at the plasma membrane is maintained at the expense of the ER/trans Golgi and nuclear membrane arachidonate-PI that is needed for proper functioning of the signaling events at the plasma membrane. A complex interplay of the Lands cycle at the ER likely exists that populates arachidonate into all phospholipid classes with the arachidonate-PI cycle, PIP and PIP2 at the plasma membrane, and PI(18:0/20:4) within the nuclear membrane of the cell (Fig. 4). Therefore, when the plasma membrane becomes depleted of PIP and PIP2 as a result of sustained signaling events at that site, the nuclear membrane and ER supply necessary PI (18:0/20:4). This could reduce the capacity of this cell to synthesize leukotrienes at the nuclear membrane. Alternatively, with sufficient and even excess arachidonate, when the Lands pathway is inhibited, much more substrate arachidonate is available at the nuclear membrane to drive leukotriene biosynthesis. Because the lowest abundance of arachidonate-PI found at subcellular locations is at the nuclear membrane, this one site would likely be the most sensitive to depletion. Regulation of the production of LTC4 by operation of the complex interaction of the AA remodeling cycle in the nuclear membrane/ER (Lands pathway) and the second messenger PI cycle located in the plasma membrane/ER suggests a very important interaction between the LPCATs and the leukotriene biosynthetic machine as a result of the subcellular location of individual molecular species of phospholipids and corresponding enzymes and transport proteins. The authors acknowledge the seminal experiments of Drs. Simona Zarini, Miguel Gijón, and Sarah Martin that have led to the ideas outlined here. Download .pdf (.08 MB) Help with pdf files arachidonic acid. ACSL, acyl CoA synthase acyl CoA synthase cytosolic phospholipase A2 inositol trisphosphate lipoxygenase lysophospholipid acyltransferase leukotriene phosphatidylinositol PI-4,5P2