Lysophospholipid acyltransferases: Novel potential regulators of the inflammatory response and target for new drug discovery

Pharmacology & Therapeutics 119 (2008) 104–114

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Pharmacology & Therapeutics j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h a r m t h e r a

Associate editor: M. Madhani

Lysophospholipid acyltransferases: Novel potential regulators of the inflammatory response and target for new drug discovery Simon K. Jackson a,⁎, Wondwossen Abate a, Amanda J. Tonks b a b

Centre for Research in Biomedicine, Faculty of Health and Life Sciences, Frenchay Campus, University of the West of England, Bristol, UK Department of Medical Microbiology, School of Medicine, Cardiff University, Heath Park, Cardiff, UK

A R T I C L E

I N F O

Keywords: Lysophospholipid Acyltransferase Lipopolysaccharide Inflammation Sepsis Monocyte Abbreviations: AA, arachidonic acid CI-976, 2,2-methyl-N-(2,4,6-trimethoxyphenyl) dodecanamide CoA, Coenzyme A CoA-IT, CoA-independent transacylase EPA, eicosapentaenoic acid FFA, free fatty acid HETP, 5 hydroxyethyl 5,3' thiophenyl pyridine IFN, interferon LPAAT, lysophosphatidic acid acyltransferase LPAT, lysophospholipid acyltransferase LPCAT, lysophoshatidylcholine acyltransferase LPS, lipopolysaccharide LBP, LPS binding protein LysoPC, lysophospahtidylcholine NFκB, nuclear factor kappa B OxLDL, oxidized low-density lipoprotein OxPL, oxidized phospholipid PAMP, pathogen-associated molecular pattern PC, phosphatidylcholine PE, phosphatidylethanolamine PL, phospholipid PLA, phospholipase PKC, protein kinase C TAZ, tafazzin TLR4, Toll-like receptor 4 TNF, tumour necrosis factor

A B S T R A C T Molecular and biochemical analyses of membrane phospholipids have revealed that, in addition to their physico-chemical properties, the metabolites of phospholipids play a crucial role in the recognition, signalling and responses of cells to a variety of stimuli. Such responses are mediated in large part by the removal and/or addition of different acyl chains to provide different phospholipid molecular species. The reacylation reactions, catalysed by specific acyltransferases control phospholipid composition and the availability of the important mediators free arachidonic acid and lysophospholipids. Lysophospholipid acyltransferases are therefore key control points for cellular responses to a variety of stimuli including inflammation. Regulation or manipulation of lysophospholipid acyltransferases may thus provide important mechanisms for novel anti-inflammatory therapies. This review will highlight mammalian lysophospholipid acyltransferases with particular reference to the potential role of lysophosphatidylcholine acyltransferase and its substrates in sepsis and other inflammatory conditions and as a potential target for novel antiinflammatory therapies. © 2008 Elsevier Inc. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (S.K. Jackson). 0163-7258/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2008.04.001

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Contents

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Acyltransferases and membrane composition . . . . . . . . . . . . 2. Characterisation of acyltransferases in mammalian cells . . . . . . . . . . 2.1. LPAT in platelets . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. LPAT in lymphocytes . . . . . . . . . . . . . . . . . . . . . . . 2.3. LPAT in phagocytic cells . . . . . . . . . . . . . . . . . . . . . 3. LysoPC and inflammation . . . . . . . . . . . . . . . . . . . . . . . . 4. Oxidized phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . 5. Phospholipid metabolites and apoptosis . . . . . . . . . . . . . . . . . 6. Phospholipid metabolism in clinical and experimental models . . . . . . . 6.1. Phospholipid metabolites and brain injury . . . . . . . . . . . . . 6.2. Barth syndrome . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Lipopolysaccharide-induced inflammatory responses in monocytes . . . . . 7.1. Priming for LPS responses in monocytes . . . . . . . . . . . . . . 7.2. Acyltransferase activity in the priming of monocytes by interferon-γ 8. Cloning and expression of LPAT . . . . . . . . . . . . . . . . . . . . . 8.1. LPAAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. LPCAT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. LPCAT as a novel target for anti-sepsis therapies . . . . . . . . . . . . . 10. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Acyltransferases and membrane composition Molecular and biochemical analyses of membrane phospholipids have revealed that, in addition to their physico-chemical properties, the metabolites of phospholipids play a crucial role in the recognition, signalling and responses of cells to a variety of stimuli. Such responses are mediated in large part by the removal and/or addition of different acyl chains to provide different phospholipid molecular species. An important phospholipid component of many membranes is phosphatidylcholine (PC) and structural studies have revealed that saturated fatty acids are usually esterified at position C1 and unsaturated fatty acids at C2 of PC (Choy & Arthur, 1989). Investigations revealed that the distribution of fatty acids did not result from the de novo biosynthesis of PC but rather from the remodelling of newly synthesised PC (Arthur & Choy, 1984). Mammalian cells and tissues contain over 100 different phospholipid molecular species. Several enzymes including acyl-CoA: lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems and lysophospholipases/transacylases are involved in the biosynthesis of these molecular species. The CoAdependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. Acyl-CoA:1-acyl-2-lysophospholipid acyltransferase (LPAT) has a preference for polyunsaturated fatty acyl-CoAs (particularly C18:1, C18:2, C20:4). The acyl-CoA: lysophospholipid acyltransferase system is thus involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids and in particular ether-containing lysophospholipids, in the absence of any cofactors and may be involved in the removal of deleterious ether-containing lysophospholipids (Yamashita et al., 1997). Such PC remodelling via deacylation–reacylation reactions is an important mechanism for the selectivity of acyl groups in mammalian tissues. The pathway for the remodelling of PC was first identified by Lands and involves the deacylation of PC to a lyso-PC and its subsequent

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reacylation back to PC with different acyl chain composition (Lands, 1960). This acylation–deacylation of membrane phospholipids (the Lands Cycle) is a mechanism for incorporating unsaturated fatty acids (mainly arachidonic acid) into different phospholipids to provide a range of lipid mediators (Yamashita et al., 1997). The fatty acids are cleaved from phospholipids by the action of phospholipase A2 and re-incorporated by acyltransferases. Even in activated cells, the reacylation reaction is significant and only a minor fraction of the free arachidonic acid is converted into eicosanoids, the remainder being reincorporated into phospholipids (Lands, 2000; MacDonald & Sprecher 1989). Studies have revealed that arachidonic-acid is incorporated first into phospholipids containing a 1-acyl linkage by Coenzyme A (CoA)-dependent enzymes. The arachidonic acid is then transferred by CoA-independent transacylases from 1-acyl linked phospholipids to 1-alkyl and 1-alk-1-enyl lysophospholipids to form 1-alkyl and 1-alk-1-enyl-2-arachidonyl phospholipids (Chilton et al.,1996), which are important in the synthesis of platelet activating factor (PAF). The reacylation of lysoPC to PC is catalysed by the action of lysoPC: acyl-CoA acyltransferase (EC 2.3.1.23, 1-acylglycerophosphocholine acyltransferase, LPCAT) in the following reaction: LysoPC þ acyl  CoA→PC þ CoA  SH LPCAT thus plays a significant role in the remodelling of membrane PC. The enzyme was first described in rat liver microsomes (Lands, 1960) and it has since been identified in a diversity of species ranging from bacteria (Proulx & Van Deenen, 1966), plants (Devor & Mudd, 1971), insects (Heckman et al., 1977) and fish (Holub et al., 1976) to mammals (Choy & Arthur, 1989). Although most of the enzyme activity in mammals is in the microsomal fraction, significant activity is also detected in the mitochondria and plasma membrane fractions (Arthur et al., 1987). These studies also suggest that a family of proteins is involved in the acylation of different lysophospholipids in different tissues. Despite the importance of LPCAT activity in controlling PC acylation, difficulty in solubilising the protein has previously hampered the isolation and characterisation of LPCAT. However, an active LPCAT enzyme has been isolated from newly-formed PC vesicles in rat liver microsomes (Fyrst et al., 1996). This approach has been used to obtain preliminary characterisation and sequence data on human

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monocyte LPCAT. Recently, two groups have identified and characterised LPCAT from alveolar type II cells in mouse (Nakanishi et al., 2006) and human (Chen et al., 2006) lungs. This enzyme might play a critically important role in the regulation of surfactant phospholipid biosynthesis, which is rich in dipalmitoyl (16:0) PC. 2. Characterisation of acyltransferases in mammalian cells Progress in the purification of LPAT has been impeded due to the instability of the enzyme and its sensitivity to detergents. Therefore most work characterising the enzyme activity and substrate preferences have been conducted on crude preparations of the enzyme from different tissues. LPAT activity has been demonstrated in nearly every human cell type. Studies indicate that there are separate acyltransferases with different specificities for the lysoPL acceptors and different acylCoAs in different tissues (Wise et al., 1980; Masuzawa et al., 1989; Ross & Kish, 1994). Interestingly, LPCAT in liver microsomes was described with a high specificity for unsaturated fatty acyl CoA (Landset al., 1982) whereas in lung microsomes it showed a preference for palmitoyl-CoA rather than unsaturated acyl CoAs (Crecelius & Longmore, 1984). This latter reflects the importance of LPCAT in lung for the synthesis of dipalmitoylPC, the major PL component of pulmonary surfactant. Moreover, experiments utilising injection of radioactive stereochemical isomers of (palmitoyl)-lysoPC into mouse lungs revealed that LPCAT but not the LysoPC:LysoPC transacylase enzyme was found to be important in the biosynthesis of di-palmitoylPC in the lung (Van Heusden et al., 1980). An investigation of acyl-CoA:lysophospholipid acyltransferase (LPAT) from crude membranes of pig spleen found the highest specificity toward 1-acyl-PC and the enzyme was able to distinguish between the acyl-chain length of the 1-acyl group within the 1-acyl-PC molecule with preference in the order C10:0 b C12:0 bbC14:0, C18:0, C16:0 b C18:1 of 1-acyl-PC. Moreover, lysophosphatidic acid (LPA) and 1-O-alkyl-PC were only poor substrates for the enzyme. The finding that palmitoyl-CoA was a poor substrate as well as an inhibitor of the enzyme was used in protein purification utilising palmitoyl-CoAagarose affinity chromatography. LAT enzyme activity was bound and eluted by high salt concentrations yielding an estimated 10-fold purification of the solubilized LAT enzyme (Kerkhoff et al., 1998). LPCAT activity in bovine retina outer rod segments was found to be maximal at pH 7.0 and the enzyme was able to incorporate 60% of [14C] oleoyl-CoA into PC after 5 min of incubation. An apparent K(m) value for oleoyl-CoA of 100 μM and a Vmax value of 153 nmol h− 1 mg protein− 1 and for lysoPC of 27 μM and a Vmax value of 155 nmol h− 1 mg protein− 1. The acyltransferase was able to incorporate other acylCoAs (palmitoyl-CoA and arachidonoyl-CoA) into rod outer segment PL and to acylate other lysoPL but less efficiently than lysoPC. LysoPC was preferentially acylated with arachidonic acid followed by oleic acid and, less efficiently, with palmitic acid (Castagnet & Giusto, 1997). 2.1. LPAT in platelets Studies on phospholipids biosynthesis in human platelets described an LPCAT activity (McKean et al., 1982). Further characterisation of this enzyme by Bakken & Farstad (1992) revealed that LPCAT had the highest affinity for linoleic acid (C18:2) as substrate followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 109 platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost exclusively (Bakken & Farstad, 1992). 2.2. LPAT in lymphocytes One of the earliest changes observed in activated lymphocytes is the enhanced incorporation of unsaturated fatty acids into membrane

phospholipids catalyzed by phospholipases and acyltransferases. Previous studies showed that this early membrane phospholipid remodelling was independent from protein synthesis but that lysophospholipidacyl-CoA acyltransferase (LAT) gene transcription is induced as an early event following T-cell activation (Goppelt-Strube & Resch, 1987). Studies of human peripheral blood lymphocytes activated with antiTCR/CD3 mAb or by dioctanoylglycerol (DiC8) and ionomycin showed both treatments induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane but IL-2 synthesis was only induced by Anti-TCR/CD3 antibodies (Szamel et al., 1989). The difference was found to be due to a secondary longer lasting activation of protein kinase C (PKC) in the antiTCR/CD3 mAb activated cells. However, addition of polyunsaturated fatty acids to the DiC8 + ionomycin-treated cells could restore IL-2 synthesis. This indicated that elevated incorporation of polyunsaturated fatty acids and thus continuous activation and translocation of PKC represents a necessary early signal for IL-2 synthesis and proliferation in human lymphocytes. The membrane phospholipid alteration induced by mitogens was found to be diminished by hydrocortisone and the inhibitory activity of hydrocortisone was specific for fatty acid incorporation into phospholipids catalyzed by lysophospholipidacyl-CoA acyltransferase (LAT) (Kerkhoff et al., 1997). Combining these observations, suggested that during T-cell activation a PC-specific PLC can provide diacylglycerol (DAG) necessary for the long-lasting activation of PKC. In this model, LAT is utilised to catalyse the enhanced incorporation of polyunsaturated fatty acids into PC which act as the precursors of DAG with the PKC-activating properties (Szamel et al., 1998). 2.3. LPAT in phagocytic cells Stimulation of human neutrophils with the ionophore A23187 induced a rapid phospholipid remodelling mediated in part by stimulated LPCAT activity (Reinhold et al., 1989). LPCAT activity was also shown to be important in a study on the characterisation of lysophospholipid metabolizing enzymes in human brain (Ross & Kish, 1994). Both CoA-dependent acyltransferase and CoA-independent transacylase (CoA-IT) activities have been demonstrated in monocytic cells (Winkler et al., 1991). This group also provided the first evidence of modulation of CoA-IT activity by a pro-inflammatory cytokine (TNFα) in human neutrophils and suggested that one mechanism for augmented lipid mediator formation is through increases in CoA-IT activity (Winkler et al., 1994). Two inhibitors of the CoAIT enzyme, SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazol1-yl)heptane-phosphonate) and SK&F 45905 [2(-)[3-(4-chloro-3trifluoromethylphenyl)ureido]-4-trifluoromethyl phenoxy]-4,5dichlorobenzenesulfonic acid) were used to further characterise the role of CoA-IT in the production of lipid mediators in human neutrophils (Winkler et al., 1995). Both inhibitors were able to decrease prostaglandin production in several inflammatory cells and to block signs of inflammation in ears of phorbol ester-challenged mice. These results showed that blockade of CoA-IT, which leads to inhibition of arachidonate remodelling between phospholipids, results in the attenuation of platelet-activating factor production, arachidonic acid release and the formation of eicosanoid products. 3. LysoPC and inflammation Lysophospholipids are the key acceptor substrates for CoAdependent acyltransferases. Lysophosphatidylcholine (lysoPC), generated by the action of phospholipase A2 on membrane phosphatidylcholine, the most abundant cellular phospholipid, is associated with a variety of physiological and pathological processes including inflammation and atherosclerosis (Steinberg et al., 1989; Yuan et al., 1996). LysoPC and LysoPA have been associated with a range of biological activities from vascular development to myelination (Birgbauer &

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Chun, 2006) and considerable effort is under way to understand the metabolism of these lipids. Utilisation of lysoPL by acyltransferases is expected to be important in the regulation of their availability and hence activity. In vitro, lysoPC has pro-inflammatory properties, as it upregulates the expression of adhesion molecules and is a chemoattractant to monocytes and T lymphocytes (Ryborg et al., 1994). It upregulates the expression of a variety of genes including genes encoding growth factors and cyclooxygenase-2 and modulates other cellular responses like proliferation and differentiation (Zembowicz et al., 1995). A role for lysoPC as an intracellular messenger transducing signals from membrane-associated receptors has also been suggested. However, the mechanisms behind the diverse actions of lysoPC are poorly understood. A recent study found that lysoPC in non-toxic concentrations caused increased activator protein-1 (AP-1) DNA-binding activity and transglutaminase-1 expression in cultured human keratinocytes. The effects on transglutaminase-1 and AP-1 were dependent on protein kinase C and mitogen-activated protein kinase kinase. In addition, lysoPC caused a rapid and transient increase in DNA-binding activity of nuclear factor-kappaB (NFκB) (Ryborg et al., 2004). A diverse range of lipid oxidation products detected in oxidized low-density lipoprotein (oxLDL) and atherosclerotic lesions are capable of eliciting biological responses in vascular cells. Experiments utilising cDNA microarray analysis showed that the lipid oxidation products oxLDL, lysophosphatidylcholine (LysoPC), 4-hydroxy-2nonenal, and oxysterols altered gene expression in HUVECs. In particular, LysoPC increased membrane protein levels of the L-type amino acid transporter 1 (LAT1) and also uptake of L-[(14)C]leucine, which was inhibited by a competitive inhibitor for LAT1. The release of interleukin 6 (IL-6) and IL-8 was increased in LysoPC-treated cells and was attenuated by the LAT1 inhibitor. These findings suggest that an increase in uptake of neutral amino acids induced by LysoPC results in enhancement of inflammatory responses of endothelial cells (Takabe et al., 2004). Lysophosphatidylcholine (LysoPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. LysoPC has the ability to initiate or amplify several steps in atherogenesis due to its ability to impair endothelium-dependent vasorelaxation, enhance endothelial proliferation and permeability, stimulate adhesion and activation of lymphocytes, initiate chemotaxis of macrophages, impair migration and proliferation in vascular smooth muscle cells (SMCs), and modify platelet aggregation and coagulation pathways (Kougias et al., 2006). Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodelling that is characteristic of atherogenesis. Recent studies have examined the role of LysoPC in this process (Aiyar et al., 2007). In these experiments, using coronary artery smooth muscle cells (CASMCs), LysoPC was found to stimulate time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. In addition, LysoPC stimulated the release of the arachidonic acid metabolites leukotriene-B4 and 6-ketoprostaglandin F1alpha, and was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. This implies that LysoPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes. Lysophospholipids are known to signal through a family of diverse G protein-coupled receptors (GPCRs). Virtually all cells that participate in the immune response express multiple receptors for LPLs. The development of antibody reagents that recognize the receptors for each LPL and the derivation of receptor-selective agonists and receptor-null mouse strains have provided insights into the widely diverse functions of LPLs in immune responses (Lin & Boyce, 2006).

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4. Oxidized phospholipids Lipid oxidation products and in particular oxidized phospholipids (OxPL) are increasingly recognized as inducers of chronic inflammation characteristic of atherosclerosis. Lysophosphatidylcholine (LysoPC) is the major bioactive lipid component of oxidized LDL, and associated with many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. OxPL stimulate production of chemokines and adhesion of monocytes to endothelial cells. However, accumulating data suggest that, in addition to the proatherogenic and pro-inflammatory effects, OxPL can stimulate anti-inflammatory and tissue-protective mechanisms. Thus, depending on the biological situation, OxPL can either stimulate or inhibit inflammation (Bochkov, 2007). Elevated plasma levels of low-density lipoprotein and generation of oxidized low-density lipoprotein have been directly associated with the pathogenesis of atherosclerosis, and lipid oxidation products have been directly linked with induction and propagation of monocytic sub endothelial accumulation and other inflammatory reactions associated with chronic vascular inflammation. However, accumulating data suggest that oxidized lipids may also exhibit anti-inflammatory potential and serve as potent inhibitors of NFκB-dependent proinflammatory cascade (Birukov, 2006). 5. Phospholipid metabolites and apoptosis Under physiological conditions apoptotic cells are engulfed by macrophages that secrete anti-inflammatory molecules or are cleared neutrally in very early phases of apoptosis. In contrast, if cells die of necrosis, a pro-inflammatory immune response is induced. Apoptotic cells that are not cleared/engulfed by phagocytes may become secondarily necrotic. Inefficient clearance of apoptotic material has been described for patients with autoimmune diseases. Thus, one current hypothesis for the pathogenesis of chronic inflammation and autoimmunity is that disturbances in the clearance of apoptotic material may lead to secondary necrosis, which in turn induces proinflammatory responses instigating and/or perpetuating autoimmune processes (Gaipl et al., 2005). An important trigger of phagocytosis of apoptotic cells phosphatidylserine (PS) exposed on the surfaces of dying cells (Savill & Fadok, 2000). In addition, the release of lysoPC has been identified as an attraction signal for apoptotic cells. LysoPC production in apoptotic cells has been shown to be triggered by the caspase 3 mediated cleavage and activation of the calcium independent phospholipase A2 (iPLA2) (Lauber et al., 2003). Factors determining the availability and release of LysoPC will therefore be important in the regulation of the clearance of apoptotic cells (Mueller et al., 2007). In unstimulated cells, the lysoPL acyltransferase-driven reacylation pathway predominates and AA levels are kept low. When cell stimulation occurs, agonist-activated PLA2 induces accumulation of AA which is then available for eicosanoid synthesis. However, AA reacylation is still significant in stimulated cells. The acyltransferase activity initially incorporates AA into PC from where it is slowly transferred by CoA-independent transacylases to other lysoPL such as 1-alkenyl-2-lysoPE (Chilton et al., 1996; Yamashita et al., 1997). In recent experiments, blocking the incorporation of AA into phospholipids has been shown to induce apoptosis in a promonocytic cell line. To manipulate the intracellular AA level in U937 phagocytes they used inhibitors of the AA reacylation pathway, namely thimerosal and triacsin C, which block the conversion of AA into arachidonoyl-CoA, a CoA-independent transacylase inhibitor that blocks the movement of AA within phospholipids and cells overexpressing group VIA phospholipase A(2), that controls basal fatty acid deacylation reactions in phagocytic cells. All of these different strategies resulted in the expected increase of cellular free AA but also in the induction of cell death by apoptosis (Perez et al., 2006). These experiments suggest that

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free AA levels within cells is an important cellular signal for apoptosis and that inhibition of the reacylation pathway controlled by LPCAT and CoAIT may be key to controlling cell survival. This may in turn lead to the development of new classes of drug aimed at controlling inflammatory cell death. 6. Phospholipid metabolism in clinical and experimental models The regulation of free fatty acids and lysophospholipids by acyltransferases has been associated with many clinical conditions. In particular, the ratio of PC/LysoPC in plasma have been found to be a reliable measure of inflammation in rheumatoid arthritis and possibly other inflammatory conditions (Fuchs et al., 2005). 6.1. Phospholipid metabolites and brain injury The deacylation–reacylation cycle is an important mechanism responsible for the introduction of polyunsaturated fatty acids into neural membrane glycerophospholipids. It involves four enzymes, namely acyl-CoA synthetase, acyl-CoA hydrolase, acyl-CoA: lysophospholipid acyltransferase, and phospholipase A2. All of these enzymes have been purified and characterised from brain tissue. Under normal conditions, the stimulation of neural membrane receptors by neurotransmitters and growth factors results in the release of arachidonic acid from neural membrane glycerophospholipids. The released arachidonic acid acts as a second messenger itself. It can be further metabolized to eicosanoids, a group of second messengers involved in a variety of neurochemical functions. A lysophospholipid, the second product of reactions catalyzed by phospholipase A2, is rapidly acylated with acyl-CoA, resulting in the maintenance of the normal and essential neural membrane glycerophospholipid composition. However, under pathological situations (ischemia), the overstimulation of phospholipase A2 results in a rapid generation and accumulation of free fatty acids including arachidonic acid, eicosanoids, and lipid peroxides. This results in neural inflammation, oxidative stress, and neurodegeneration. In neural membranes, the deacylation–reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. This is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes (Farooqui et al., 2000). Cerebral insult is associated with a rapid increase in free fatty acids (FFA) and arachidonic acid release and has been linked to the increase in eicosanoid biosynthesis. In transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion, there is an inverse relationship between the increase in FFA and the decrease in ATP, both during the period of ischemia and at later time periods after reperfusion. In a study by Zhang and Sun (1995), the focal cerebral ischemia model was used to examine incorporation of [14C]arachidonic acid into the glycerolipids in rat MCA cortex at different reperfusion times after a 60 min ischemia. Labelled arachidonic acid was incorporated into phosphatidylcholine, phosphatidylethanolamine and neutral glycerides. With increasing time (4–16 h) after a 60 min ischemia, an inhibition of labelled arachidonate uptake could be found in the right ischemic MCA cortex, whereas the distribution of radioactivity among the major phospholipids was not altered. In an in vitro assay system, synaptosomal membranes isolated from MCA cortex 8 and 16 h after a 60 min ischemia showed a significant decrease in arachidonoyl transfer to lysophospholipids, mainly due to a decrease in lysophospholipid:acylCoA acyltransferase activity. Assay of phospholipase A2 activity with both synaptosomes and cytosol, however, did not show differences between left and right MCA cortex or with time after reperfusion. These results suggest that besides ATP availability, the decrease in acyltransferase activity may also contribute to the increase in FFA in cerebral ischemia–reperfusion.

Damage to brain membrane phospholipids may play an important role in the pathogenesis of Alzheimer's disease (AD); however, the critical metabolic processes responsible for the generation and repair of membrane phospholipids affected by the disease are unknown. In a study measuring the activity of key phospholipid catabolic and anabolic enzymes in AD the activity of the major catabolic enzyme phospholipase A2 (PLA2) was significantly decreased (−35 to −53%) in parietal and temporal cortices of patients with AD (Ross et al., 1998). In contrast, the activities of lysophospholipid acyltransferase, which recycles lysophospholipids into intact phospholipids, and glycerophosphocholine phosphodiesterase, which returns phospholipid catabolites to be used in phospholipid resynthesis, were increased by approximately 50–70% in the same brain areas. Furthermore, the activities of PLA2 and acyltransferase were normal in the degenerating cerebellum of patients with spinocerebellar atrophy type 1, whereas the activity of glycerophosphocholine phosphodiesterase was reduced, suggesting that the alterations in AD brain were not nonspecific consequences of neurodegeneration. 6.2. Barth syndrome Barth Syndrome is an X-linked recessive genetic disorder characterised by cardiomyopathy and neutropenia. The disorder is associated with abnormal mitochondria and respiratory chain dysfunction (Barth et al., 1983). Tafazzin (TAZ) has been identified as the gene responsible for the condition (Neuwald, 1997), which had led to the development of improved diagnostic tests for the condition. The condition is devastating, often proving fatal in infancy or early childhood as a result of severe bacterial infections or cardiac failure. Understanding the underlying causes of the disease and the pathological mechanisms which underlie the condition is the best hope of developing treatments to improve quality of life and increase life expectancy for individuals affected by this disease (Barth et al., 2004). The TAZ gene is located on region q28 of the X chromosome, has significant protein homology to other acyltransferase family members and is believed to affect lipid metabolism, specifically related to Cardiolipin. Cardiolipin levels are depressed in patients with Barth syndrome (Vreken et al., 2000). This lipid moiety is found mainly in mitochondrial membranes, and is believed to stabilise cellular membranes (Koshkin & Greenberg, 2002) and/or aid functionality of membrane associated proteins (Schlame et al., 2000). Reduced cardiolipin levels or altered acyl content may contribute to the pathology of Barth syndrome by affecting respiratory chain complexes and function (Brandner et al., 2005). 6.3. Sepsis Sepsis is a consequence of an overwhelming inflammatory response to infection which has proved exceptionally difficult to treat despite advances in antibiotic therapy and intensive care. Sepsis occurs in at least one and a half million people throughout the world each year. In the United States alone, sepsis develops in more than 500,000 patients each year, with a 30–70% mortality rate (Angus et al., 2006). Sepsis develops from the systemic inflammatory response to pathogens in the blood. Bacterial pathogens carry surface molecules termed pathogen associated molecular patterns (PAMPs) which can induce a variety of inflammatory mediators. The resulting inflammatory response can produce cardiovascular derangements, hypotension, multiple organ failure and death (Cohen, 2002). The best characterised of these microbial PAMP is the bacterial molecule lipopolysaccharide (LPS), the endotoxin present in the outer membrane of Gram-negative bacteria (Rietschel et al., 1994; Van Amersfoot et al., 2003). Key target cells in the pathogenesis of LPS-induced sepsis are the monocytes and macrophages.

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7. Lipopolysaccharide-induced inflammatory responses in monocytes Monocytes and macrophages can respond to pg amounts of LPS in the circulation by the production of inflammatory mediators including cytokines and bioactive lipids, and the expression of cell-surface receptors and adhesion molecules (Glauser et al., 1991; Bhatia & Moochhala, 2004). It is the massive release of the inflammatory mediators that is a primary mechanism for the initiation of severe sepsis (Hesse et al., 1988; Tracey & Lowry, 1990). Among the inflammatory cytokines expressed in response to LPS stimulation, TNFα and IFN-γ have been shown to be particularly important in the development of septic shock (Doherty et al., 1992; Silva & Cohen, 1992; Rothe et al., 1993). Recently, a later acting mediator, HMGB-1, has been shown to propagate inflammatory responses in sepsis (Wang et al., 2004). LPS induces inflammatory mediator production in monocytes and macrophages via binding to the cell surface receptor Toll-like receptor 4 (TLR4) (Akira & Takeda, 2005, 2004). LPS released in the circulation from invading bacteria, either spontaneously during growth or as a consequence of immune-mediated lysis, binds to numerous lipid binding proteins including albumin, transferrin, high density lipoproteins and LPS-binding protein (LBP), an acute phase protein synthesised by the liver in response to infection. LBP complexes LPS and acts as a lipid transfer protein shuttling LPS to the surface of monocytes and macrophages (Cohen, 2002, Schumann et al., 1990). CD14, a 55 kDa glycosylphosphoinositol (GPI)-anchored membrane protein on the surface of monocytes and macrophages (Wright et al., 1990; Pugin et al., 1994) binds to LPS transferred from LBP and in turn shuttles LPS to the signalling receptor TLR4. Soluble CD14 can also bind and transfer LPS to cell-surface receptors in cells lacking membrane CD14. A small protein, MD2, associates with TLR4 and is essential for responses to LPS (Shimazu et al., 1999) and CD14 couples with TLR4 and MD2 (Akashi et al., 2000) to form a complex that induces cell activation in response to LPS. Using biochemical and fluorescence imaging techniques, TLR4 (and TLR2) has been found to be recruited into membrane microdomains (lipid rafts) on cell stimulation with LPS (Triantafilou et al., 2002). 7.1. Priming for LPS responses in monocytes Priming for enhanced responses to LPS is of considerable interest clinically because it is thought that underlying or sub-clinical infections may prime patients for exaggerated inflammatory responses to low concentrations of LPS. However, the molecular mechanisms of this priming are not well understood. IFN-γ has been shown to be an important mediator of the sensitising actions of infectious agents, such as P. acnes, on macrophages for LPS (Katschinski et al., 1992; Billiau et al., 1987). Indeed, a major contribution of IFN-γ to LPS-induced shock may be priming an enhanced activation state in monocytes/macrophages (Silva & Cohen 1992). Upon subsequent exposure to LPS, the primed macrophages become hyper-activated and produce excessive inflammatory mediators including TNFα and IL-1β (Heinzel, 1990; Doherty et al., 1992). Changes in the functional and plasma membrane organizational states of human neutrophils after stimulation with known priming agents (LPS, TNF-alpha, or GM-CSF) suggest that priming of suspended, circulating neutrophils is associated with a large-scale reorganization of the plasma membrane and associated membrane cortex in a process that is independent of cellular adhesion and gross morphologic polarization (Stie & Jesaitis, 2007). 7.2. Acyltransferase activity in the priming of monocytes by interferon-γ The precise mechanisms underlying the priming of macrophages by INF-γ for enhanced responses to LPS have remained elusive. Recent

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work has suggested that IFN-γ might increase responsiveness to LPS by augmenting the signal transduction pathway including upregulating TLR4 expression (Bosisio, 2002) or promoting IL-1 receptor associated kinase expression and its association to MyD88 (Adib-Conquy & Cavaillon 1992). Our laboratory has been concerned with elucidating the mechanisms of the priming responses of monocytes and macrophages to LPS. We have previously established that infections, such as BCG, which increase sensitivity to LPS in experimental models of sepsis, alter the membrane phospholipid profiles of macrophages and monocytes (Stark et al., 1990). Furthermore, these ‘priming’ infections were shown to induce the production of IFN-γ which mediated the macrophage/monocyte responsiveness. We subsequently showed that IFN-γ could produce similar alterations in macrophage phospholipid compositions that accompany the priming of these cells both in vivo and in vitro (Jackson et al., 1989; Jackson et al., 1993). In particular, IFN-γ stimulated the increased incorporation of unsaturated fatty acids into phosphatidylcholine (PC) which were then turned over into phosphatidylethanolamine (PE) (Darmani et al., 1993). The incorporation of unsaturated fatty acids into phospholipids is accomplished by the deacylation and reacylation of the phospholipids (the Lands Cycle) mediated by the activity of the lipid modifying enzymes phospholipases and acyltransferases (Balsinde, 2002). Thus, we suggested that IFN-γ might increase responsiveness to LPS in macrophages by upregulating the activity of these enzymes. While the activity of phospholipases was not influenced by IFN-γ, the activity of certain acyltransferases was significantly increased by this priming agent (Schmid et al., 2003). Both CoA-dependent acyltransferases and CoA-independent transacylases in addition to their important role in providing substrates for lipid mediators of inflammation, have been found to be involved in lipid signalling pathways (Jackson, 1997; Prokazova et al., 1998) and leukocyte activation. They may also modulate the activities of other membrane-localized enzymes. However, little is known about the regulation of these enzymes during inflammation, although a study using human neutrophils described a PAF-induced increase in the arachidonoyl-CoA-specific LPCAT activity (Tou, 1987). Similarly, TNF was found to increase the CoAIT activity in human neutrophils (Winkler et al., 1994) and IL-1α was shown to increase the incorporation of arachidonate into phosphatidylinositol and phosphatidylserine in rat mesangial cells, implying an enhanced rate of arachidonate-selective lysophospholipid acyltransferase(s) (Winkler et al., 1995). Studies from our laboratory recently demonstrated that TNF can modify phospholipid compositions in monocytes via activation of CoA-independent transacylases (Neville et al., 2005). Furthermore, we showed that IFN-γ and concanavalin A, another priming agent, could selectively activate LPCAT but not the lysophosphatidic acid acyltransferase (LPAAT) (Schmid et al., 2003). Thus, LPCAT activity is upregulated under conditions of priming monocytes/macrophages for increased responses to LPS. Regulation of the signalling pathway for LPS-mediated responses is a focus of research aimed at developing new therapies for sepsis and related inflammatory disease. We have demonstrated the importance of LPCAT in the responses of phagocytic cells to LPS and have shown using specific LPCAT inhibitors that this effect must be at the level of the LPS receptor complex in the plasma membrane. On cell stimulation with LPS, components of this receptor assemble in membrane lipid raft microdomains (Triantafilou et al., 2002). Our hypothesis is that membrane PC composition affects membrane fluidity and the effectiveness of functional receptor complex aggregation in lipid rafts (Jackson & Parton, 2004). Recent experiments have demonstrated that inhibition of LPCAT in monocytes can prevent the translocation of the LPS receptor TLR4 into lipid raft domains (Jackson et al., in press). This would provide a novel regulation of inflammatory responses in these cells. Development of new therapeutic approaches to sepsis and other inflammatory conditions based on LPAT inhibition will be enhanced by knowledge of the sequence data for these enzymes. Progress in the cloning and expression of both LPAAT and LPCAT is therefore outlined.

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8. Cloning and expression of LPAT 8.1. LPAAT Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. LPA acyltransferase (LPAAT), also known as 1-acyl sn-glycerol-3phosphate acetyltransferase (EC 2.3.1.51), catalyzes the conversion of LPA to PA. In one of the first descriptions of a cDNA sequence of a mammalian gene homologous to non-mammalian LPAAT, Stamps et al. (1997) describe the isolation of a human homologue of Escherichia coli, yeast and plant 1-acylglycerol-3-phosphate acyltransferase from U937 cell cDNA. Expression of the cloned sequence in 1-acylglycerol-3phosphate acyltransferase-deficient E. coli resulted in increased incorporation of oleic acid into cellular phospholipids. Membranes made from COS7 cells transfected with the cDNA exhibited higher acyltransferase activity towards a range of donor fatty acyl-CoAs and lysophosphatidic acid. Northern-blot analysis of the cDNA sequence indicated high levels of expression in immune cells and epithelium. Rapid amplification of cDNA ends revealed differentially expressed splice variants, which suggests regulation of the enzyme by alternative splicing. This cDNA therefore represents the first described. Several isoforms of LPAAT exist and have been isolated and characterised. Two proteins derived from human cDNA sequences were designated as LPAAT-alpha and LPAAT-beta, and found to contain extensive sequence similarities to microbial or plant LPAAT sequences (West et al., 1997). LPAAT-alpha mRNA was detected in all tissues with highest expression in skeletal muscle whereas LPAAT-beta was expressed predominantly in heart and liver tissues. Overexpression of these two cDNAs in mammalian cells led to increased LPAAT activity in cell-free extracts and correlated with enhancement of transcription and synthesis of tumour necrosis factor-alpha (TNFα) and interleukin6 (IL-6) from cells upon stimulation with interleukin-1beta, suggesting LPAAT over expression may amplify cellular signalling responses from cytokines. In another set of experiments, a human expressed sequence tag was identified by homology with a coconut LPAAT and used to isolate a full-length human cDNA from a heart muscle library (Eberhardt et al., 1997). Recombinant protein produced in COS 7 cells exhibited LPAAT activity with a preference for LPA as the acceptor phosphoglycerol and arachidonyl coenzyme A as the acyl donor. The human LPAAT gene was found to be contained on six exons that map to chromosome 9, region q34.3. Further work by the same group (Eberhardt et al., 1999) described a pair of human LPAAT isozymes encoded by distinct genes located on different chromosomes but sharing sequence homology, substrate specificity, and intracellular location. The biological value of maintaining the two closely related LPAAT genes in the human genome is not clear when they have the same tissue and sub-cellular localisation and similar substrate specificities.

Sequence analysis of cDNA clones corresponding to a number of genes located in the class III region of the human major histocompatibility complex (MHC), in the chromosome band 6p21.3, showed that the G15 gene encodes a 283-amino acid polypeptide with significant homology over the entire polypeptide with LPAAT from different yeast, plant, and bacterial species (Aguado & Campbell, 1998). The hLPAATalpha polypeptide expressed in the mammalian CHO cell line was found, by confocal immunofluorescence, to be localized in the endoplasmic reticulum. These authors suggested that due to the known role of LPA and PA in intracellular signalling and inflammation, the hLPAATalpha gene could represent a candidate gene for some MHC-associated diseases. The identification of a novel LPAAT member, designated as LPAATzeta, has recently been described (Li et al., 2003). LPAAT-zeta was predicted to encode a protein consisting of 456 amino acid residues with a signal peptide sequence and the acyltransferase domain. Northern blot analysis showed that LPAAT-zeta was ubiquitously expressed in all 16 human tissues examined, with levels in the skeletal muscle, heart, and testis being relatively high and in the lung being relatively low. The human LPAAT-zeta gene consisted of 13 exons and is positioned at chromosome 8p11.21. 8.2. LPCAT Acyltransferases are central to phospholipid metabolism, they play an important role in remodelling phospholipid fatty acids and protect cellular membranes from the damaging effects of lysophospholipids (Heath & Rock, 1998). In spite of this essential role and the ubiquitous expression of such enzymes, very little is known about their metabolic activity, regulation, specific gene sequences and tissue distribution. The majority of information relating to acyltransferase enzymes has been gleaned form studies utilising LPCAT enzyme activity assays and chemical inhibitors. The slow progress in acyltransferase research is largely due to the chemical nature of these enzymes and their cellular location. In general acyltransferase enzymes are tightly associated with cellular membranes which make them difficult to express in various systems and hard to purify. The hydrophobic chemical nature of the enzymes is problematic for the formation of crystal structures, which in turn hinders high resolution structural analysis by techniques such as NMR and X-ray diffraction (Welte & Wacker, 1991). A sn-glycerol-3-phosphate acyltransferase has previously been successfully purified and studied (Scheideler & Bell, 1991), however the kinetic properties of the enzyme were found to differ from the native membrane associated protein, highlighting the difficulties associated with the specific dissection of structure/function relationships for this group of enzymes. Recently lung specific acyltransferase has been identified and characterised in mice (Chen et al., 2006; Nakanishi et al., 2006). This acyltransferase is believed to play a specific role in the generation of the major constituent of pulmonary surfactant, dipalmitoyl phosphatidylcholine. Pulmonary surfactant is the lipid rich fluid which lines the

Fig. 1. Partial protein sequences for human (NP_079106), murine (NP-663351) and rat (XP_341748) LPCAT illustrating cross species homology. The domain for acyltransferase activity is highlighted.

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Fig. 2. Sequence homology observed across LPCAT (NP_079106), LPAAT (AAB47493.1) and Taffazin (NP_000107.1) sequences. Note the universal presence of the FPEGXX domain and the variable presence of the NHXSXXD domain.

alveoli and small airways. Composed of 90% lipid and 10% surfactant specific proteins, the putative function of this heterogeneous material is to lower the surface tension at the liquid–air interface and prevent alveolar collapse following expiration (Griese, 1999). Phospholipids are the predominant components of pulmonary surfactant, with phosphatidylcholine (PC) accounting for up to 70–80% of the total lipid content by weight, approximately 50% of which is in the form of dipalmitoyl phosphatidylcholine (DPPC) (Veldhuizen et al., 1998). Infant respiratory distress syndrome (IRDS) is the most common cause of acute respiratory symptoms in neonates and is due to deficient surfactant activity, usually associated with premature birth. Surfactant activity usually develops around 32–35 weeks of gestation in man (Brewis, 1980). In addition to IRDS, surfactant insufficiency and alterations in quality are observed in numerous respiratory diseases including acute respiratory distress syndrome (ARDS) (Nakos et al., 1998), allergic alveolitis (Heeley et al., 2000) and cystic fibrosis (Postle et al., 1999). Understanding the way in which surfactant production is regulated via acyltransferases may pave the way for the development of novel therapeutics. The role of acyltransferase activity in the generation of surfactant DPPC has been well established in previous metabolic labelling experiments (Den Breejen et al., 1989). Chen and co-workers identified a lung specific acyltransferase using gene expression microarray technology to assess differential gene expression in lung tissue at various stages of embryonic development (Chen et al., 2006). The identified lysoPC acyltransferase appears to catalyse the production of DPPC in type II alveolar cells, the major cellular source of pulmonary surfactant. The enzyme has a preference for palmitoyl CoA, is increasing expressed in embryonic tissue over time and expression is upregulated by Glucocorticoid steroids which have been shown to accelerate maturation of the fetal lungs and increase surfactant production. The identification of the murine and rat forms of lung specific LPCAT gene led Chen and co-workers to identify the human ortholog which has 89% sequence identity with the mouse and rat genes (Chen et al., 2006) (Illustrated in Fig. 1). The acyltransferase domain present shows greater than 99% sequence identity across the species examined. The conserved acyltransferase domain illustrated is a key feature of a family of proteins, which includes acyltransferases involved in phospholipid biosynthesis and other proteins of unknown function. This family also includes tafazzin, the Barth syndrome gene. The broad nature of this protein family is an indication of the complexity and diverse functions if this group of enzymes. Fig. 2 shows the sequence homology observed across the enzymes LPCAT, LPAAT and Tafazzin. The FPEGXX and NXSXXD motifs have been indicated to be catalytic domains in the acyltransferase domain. Although the FPEGXX region appears to remain fairly constant, the NXSXXD region may be more variable. Comparative analysis of protein localisation sites using PSORT software utilising examples from both the LPAAT and LPCAT gene families and the Taffazin family of sequences has revealed the presence of transmembrane regions, and predict localisation to cellular membranes including the plasma membrane and endoplasmic reticulum. This may account for some

of the difficulties that have previously been encountered in purification of these enzymes. 9. LPCAT as a novel target for anti-sepsis therapies Targeting signal transduction has emerged as a promising strategy to treat inflammatory diseases (O'Neill, 2006). The modulation of inflammatory gene expression by lipids, particularly through the Tolllike receptors, has encouraged the development of new classes of antiinflammatory molecules based on lipid modifications (Lee & Hwang, 2006). Two structurally diverse inhibitors of CoA-IT activity, SK&F 98625 [diethyl 7-(3,4,5-triphenyl-2-oxo-2,3-dihydro-imidazole-1-yl)heptane phosphonate] and SK&F 45905 [2-[2-(3-4-chloro-3-(trifluoromethyl)phenyl)-ureido]-4-(trifluoromethyl)phenoxy]-4,5-dichlorobenzenesulfonic acid] were developed (Chilton et al., 1995). These compounds were tested for their capacity to block microsomal CoA-IT activity using two assay systems, the transacylation of 1alkyl-2-lyso-sn-glycero-3-phosphocholine (GPC) and the transfer of [14C]arachidonate from 1-acyl-2-[14C]arachidonoyl-GPC to lyso-PE. Both SK&F 98625 and SK&F 45905 inhibited CoA-IT activity (IC50s 6– 19 μM) in these two assays. In contrast, SK&F 98625 or SK&F 45905 had little or no effect on other lipid-modifying activities, including CoA-dependent acyltransferase or acetyltransferase. Kinetic analysis revealed that both SK&F 98625 and SK&F 45905 interact directly with the enzyme and prevented the acylation of lysophospholipids in a competitive manner. In intact human neutrophils, both SK&F 98625 and SK&F 45905 completely blocked the movement of [3H]arachidonate from 1-acyl-linked phospholipids into 1-alkyl-2-arachidonoylGPC and 1-alk-1'-enyl-2-arachidonoyl-GPE. In contrast, these compounds did not inhibit the incorporation of free arachidonic acid into cellular lipids indicating that they did not alter CoA-dependent acyl transferase activities in the intact cell. Understanding and modulating the production of LPS-induced mediators such as TNFα has been the focus of much research aimed at developing specific therapies for septic shock. The potential role of LPCAT inhibition in the treatment of sepsis and potentially other inflammatory conditions is highlighted by experiments demonstrating the effectiveness in vitro and studies showing the therapeutic effects of lysoPA and lysoPC in experimental models of sepsis and organ failure (Murch et al., 2006, 2007; Yan et al., 2004). In experiments to test the hypothesis that oxidized 1-palmitoyl-2arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) may attenuate the acute lung inflammatory response to lipopolysaccharide (LPS) and enhance lung vascular barrier recovery in vivo and in vitro models of LPS-induced lung injury were used (Nonas et al., 2006). In vivo, aerosolized intratracheal LPS induced lung injury with profound increases in bronchoalveolar lavage neutrophils, protein content, and the inflammatory cytokines interleukin 6 and interleukin 1beta, as well as tissue neutrophils. OxPAPC, but not nonoxidized PAPC, markedly attenuated the LPS-induced tissue inflammation, barrier disruption, and cytokine production over a range of doses.

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The difficulty in purifying LPATs and a lack of sequence data for the LPCAT enzymes, have hampered development of specific small molecule inhibitors. However, recent studies have identified a novel lysophospholipid acyltransferase (LPAT) that is associated with the Golgi complex and that is sensitive to the previously characterised acyl-CoA cholesterol acyltransferase inhibitor, 2,2-methyl-N-(2,4,6trimethoxyphenyl)dodecanamide (CI-976) (Chambers and Brown, 2004). Studies utilising this inhibitor demonstrate that CI-976 inhibits multiple membrane trafficking steps, including ones found in the endocytic and secretory pathways, and imply a wider role for lysophospholipid acyltransferases in membrane trafficking (Chambers et al., 2005). A recent review describes the use of acyltransferase inhibitors as probes for studying the potential role of lysophospholipid acyltransferases (LPAT) in intracellular membrane trafficking in the secretory and endocytic pathways (Brown & Schmidt, 2005). In our laboratories, we have used selective inhibitors for LPCAT identified by high-throughput screening. A promising candidate that has emerged is 5 hydroxyethyl 5,3′ thiophenyl pyridine (HETP), a noncompetitive specific inhibitor of CoA-dependent LPCAT (Schmid et al., 2003) which was synthesised by a method adapted from Yamada et al. (2005). In the cell systems used, HETP was found to have an IC50 of 10 μM for the inhibition of LPCAT. We have recently identified a cDNA sequence for LPCAT from human monocytes and are using this sequence to develop inhibitory RNA sequences as potential LPCAT inhibitors. 10. Conclusions In summary, acyltransferases and in particular, LPCAT, have emerged as novel potential regulators of inflammatory responses in human cells. These enzymes thus provide novel targets for the development of new anti-inflammatory drugs aimed at controlling the phospholipid/lysophospholipid balance. In addition, they offer, for the first time, the possibility of specific therapies for the overwhelming inflammatory responses of sepsis. References Adib-Conquy, M., & Cavaillon, J. M. (1992). Gamma interferon and granulocyte/ monocyte colony-stimulating factor prevent endotoxin tolerance in human monocytes by promoting interleukin-1 receptor associated kinase expression and its association to MyD88 and not by modulating TLR4 expression. J Biol Chem 277, 27927−27934. Aguado, B., & Campbell, R. D. (1998). Characterization of a human lysophosphatidic acid acyltransferase that is encoded by a gene located in the class III region of the human major histocompatibility complex. J Biol Chem 273, 4096−4105. Aiyar, N., Disa, J., Ao, Z., Ju, H., Nerurkar, S., Willette, R. N., Macphee, C. H., et al. (2007). Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells. Mol Cell Biochem 295, 113−120. Akashi, S., Shimazu, R., Ogata, H., Nagai, Y., Takeda, K., Kimoto, M., et al. (2000). Cell surface expression and lipopolysaccharide signalling via the Toll-lioke receptor 4MD2 complex on mouse peritoneal macrophages. J Immunol 164, 3471−3475. Akira, S., & Takeda, K. (2004). Toll-like receptor signalling. Nat Rev Immunol 4, 499−511. Akira, S., & Takeda, K. (2005). Pathogen recognition with Toll-like receptors. Curr Opinion Immunol 17, 338−344. Angus, D. C., Pereira, C. A., & Silva, E. (2006). Epidemiology of severe sepsis around the world. Endocr Metab Immune Disord Drug Targets 6, 207−212. Arthur, G., & Choy, P. C. (1984). Acyl specificity of hamster heart CDP-choline 1,2diacylglycerol phosphocholine transferase in phosphatidylcholine biosynthesis. Biochim Biophys Acta 795, 221−229. Arthur, G., Page, L. L., Zaborniak, C. L., & Choy, P. C. (1987). The acylation of lysophosphoradylglycerocholines in guinea-pig heart mitochondria. Biochem J 242, 171−175. Bakken, A. M., & Farstad, M. (1992). The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets. Biochem J 288, 763−770. Barth, P. G., Scholte, H. R., Berden, J. A., van der klei-van Moorsel, J. M., Luyt-Houwen, I. E., van't veer-korthof, E. T., et al. (1983). An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophils leucocytes. J Neurol Sci 364, 327−355. Balsinde, J. (2002). Roles of various phospholipases A2 in providing lysophospholipid acceptors for fatty acid phospholipids incorporation and remodelling. Biocem J 364, 695−702. Barth, P. G., Valianpour, F., Bowen, V. M., Lam, J., Duran, M., Vaz, F. M., et al. (2004). X-linked cardioskeletal myopathy and neutropenia (Barth Syndrome): An update. Am J Med Genet 126, 349−354.

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