Phospholipase A2 structure/function, mechanism, and signaling
2008; Elsevier BV; Volume: 50; Linguagem: Inglês
10.1194/jlr.r800033-jlr200
ISSN1539-7262
AutoresJohn E. Burke, Edward A. Dennis,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoTremendous advances in understanding the structure and function of the superfamily of phospholipase A2 (PLA2) enzymes has occurred in the twenty-first century. The superfamily includes 15 groups comprising four main types including the secreted sPLA2, cytosolic cPLA2, calcium-independent iPLA2, and platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated (Lp)PLA2. We review herein our current understanding of the structure and interaction with substrate phospholipids, which resides in membranes for a representative of each of these main types of PLA2. We will also briefly review the development of inhibitors of these enzymes and their roles in lipid signaling. Tremendous advances in understanding the structure and function of the superfamily of phospholipase A2 (PLA2) enzymes has occurred in the twenty-first century. The superfamily includes 15 groups comprising four main types including the secreted sPLA2, cytosolic cPLA2, calcium-independent iPLA2, and platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated (Lp)PLA2. We review herein our current understanding of the structure and interaction with substrate phospholipids, which resides in membranes for a representative of each of these main types of PLA2. We will also briefly review the development of inhibitors of these enzymes and their roles in lipid signaling. The last 25 years has witnessed a virtual explosion in our knowledge about the superfamily of phospholipase A2 (PLA2) enzymes. PLA2 hydrolyzes the fatty acid from the sn-2 position of membrane phospholipids. In vivo, the sn-2 position of phospholipids frequently contains polyunsaturated fatty acids, and when released, these can be metabolized to form various eicosanoids and related bioactive lipid mediators (1Funk C.D. Prostaglandins and leukotrienes: advances in eicosanoid biology.Science. 2001; 294: 1871-1875Crossref PubMed Scopus (2997) Google Scholar). The remaining lysophospholipid can also have important roles in biological processes (2Rivera R. Chun J. Biological effects of lysophospholipids.Rev. Physiol. Biochem. Pharmacol. 2008; 160: 25-46PubMed Google Scholar).From the end of the nineteenth and beginning of the twentieth century (3Stephens W.W. Walker J.L. Myers W. The action of cobra poison on the blood: a contribution to the study of passive immunity.J. Pathol. Bacteriol. 1898; 5: 279-301Crossref Google Scholar), PLA2 was known to be a major component of snake venoms, and it was later recognized that PLA2 from old world snakes (group I) differed in their disulfide bond pattern from new world snakes (group II). Later it was discovered that the major mammalian digestive enzyme, pancreatic PLA2, was more similar to that from the old world snakes such as the Indian cobra (group IA), and hence the pancreatic enzyme was named group IB. With the isolation, sequencing, and cloning of the PLA2 from human synovial fluid in 1988 (group IIA) (4Seilhamer J.J. Pruzanski W. Vadas P. Plant S. Miller J.A. Kloss J. Johnson L.K. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid.J. Biol. Chem. 1989; 264: 5335-5338Abstract Full Text PDF PubMed Google Scholar, 5Kramer R.M. Hession C. Johansen B. Hayes G. McGray P. Chow E.P. Tizard R. Pepinsky R.B. Structure and properties of a human non-pancreatic phospholipase A2.J. Biol. Chem. 1989; 264: 5768-5775Abstract Full Text PDF PubMed Google Scholar), which had a disulfide bond pattern more similar to the new world rattlesnakes (group II), the more complicated PLA2 from bee venom (group III) (6Kuchler K. Gmachl M. Sippl M.J. Kreil G. Analysis of the Cdna for phospholipase-A2 from honeybee venom glands - the deduced amino-acid sequence reveals homology to the corresponding vertebrate enzymes.Eur. J. Biochem. 1989; 184: 249-254Crossref PubMed Scopus (152) Google Scholar), and in 1991 the human cytosolic calcium-dependent PLA2 from macrophages (group IVA) (7Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 8Kramer R.M. Roberts E.F. Manetta J. Putnam J.E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells.J. Biol. Chem. 1991; 266: 5268-5272Abstract Full Text PDF PubMed Google Scholar), the need for a more elaborate “group numbering system” became obvious (9Dennis E.A. Diversity of group types, regulation, and function of phospholipase A2.J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar). As the discovery of additional PLA2s continued such as the macrophage secreted group V PLA2 (10Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. Cloning and recombinant expression of a novel human low-molecular-weight Ca2+-dependent phospholipase-a(2).J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar, 11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. Novel group V phospholipase A2 involved in arachidonic acid mobilization in murine P388D1 macrophages.J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) and the calcium-independent PLA2 (group VI) (12Ackermann E.J. Kempner E.S. Dennis E.A. Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar), this system was expanded with 14 distinct groups and many subgroups appearing by 2000 (13Six D.A. Dennis E.A. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization.Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1203) Google Scholar). The latest review (14Schaloske R.H. Dennis E.A. The phospholipase A2 superfamily and its group numbering system.Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (720) Google Scholar) lists 15 distinct groups of PLA2. They cluster in four main categories or types: secreted sPLA2s, cytosolic cPLA2s, calcium-independent iPLA2s, and platelet activating factor (PAF) acetyl hydrolase/oxidized lipid lipoprotein associated (Lp)PLA2s. Each of these types has been implicated in diverse kinds of lipid metabolism and disease progression so there has been a tremendous interest in the pharmaceutical and biotechnology industry in developing selective and potent inhibitors of each of these types.SECRETED PLA2The secreted PLA2s were the first type of PLA2 enzymes discovered. They are found in sources as diverse as venoms from various snakes, scorpions, etc.; components of pancreatic juices; arthritic synovial fluid; and in many different mammalian tissues (13Six D.A. Dennis E.A. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization.Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1203) Google Scholar). They are characterized by a low molecular weight (13–15 kDa), histidine in the catalytic site, Ca2+ bound in the active site, as well as six conserved disulfide bonds with one or two variable disulfide bonds. These enzymes all catalyze the hydrolysis through the same mechanism of abstraction of a proton from a water molecule followed by a nucleophilic attack on the sn-2 bond. The water molecule is activated by the presence of a histidine/aspartic acid dyad in a Ca2+ dependent manner (15Scott D.L. White S.P. Otwinowski Z. Yuan W. Gelb M.H. Sigler P.B. Interfacial catalysis: the mechanism of phospholipase A2.Science. 1990; 250: 1541-1546Crossref PubMed Scopus (674) Google Scholar, 16Yu L. Dennis E.A. Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.Proc. Natl. Acad. Sci. USA. 1991; 88: 9325-9329Crossref PubMed Scopus (53) Google Scholar). Most of the secreted PLA2 enzymes share the property of exhibiting an increase in activity termed interfacial activation when substrate is presented as a large lipid aggregate, rather than in monomeric form. More detailed reviews of interfacial kinetics can be found elsewhere (17Gelb M.H. Jain M.K. Hanel A.M. Berg O.G. Interfacial enzymology of glycerolipid hydrolases: lessons from secreted phospholipases A2.Annu. Rev. Biochem. 1995; 64: 653-688Crossref PubMed Scopus (224) Google Scholar, 18Carman G.M. Deems R.A. Dennis E.A. Lipid signaling enzymes and surface dilution kinetics.J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar).Understanding the mechanism of interfacial activation as well as the orientation of lipid binding has long been a goal of mechanistic studies of the secreted PLA2s. Experiments using nuclear magnetic resonance derived nuclear overhauser effect results have been used to map the binding sites of a single phospholipid substrate in the cobra venom group IA PLA2 as shown in Fig. 1A(19Plesniak L.A. Yu L. Dennis E.A. Conformation of micellar phospholipid bound to the active site of phospholipase A2.Biochemistry. 1995; 34: 4943-4951Crossref PubMed Scopus (22) Google Scholar). Recent work using deuterium exchange mass spectrometry with phospholipid surface present has generated a model of how this same enzyme binds to the lipid surface as shown in Fig. 1B (20Burke J.E. Karbarz M.J. Deems R.A. Li S. Woods Jr., V.L. Dennis E.A. Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry.Biochemistry. 2008; 47: 6451-6459Crossref PubMed Scopus (49) Google Scholar). The group IA enzyme appears to bind lipid substrate in the active site through the hydrophobic residues lining the active site channel, and binds neutral membrane substrate through interactions with a group of hydrophobic residues on the lipid binding surface of the molecule. Experiments conducted with the group III bee venom have used electrostatic potential-modulated spin relaxation magnetic resonance to determine how that enzyme binds the lipid surface (21Lin Y. Nielsen R. Murray D. Hubbell W.L. Mailer C. Robinson B.H. Gelb M.H. Docking phospholipase A2 on membranes using electrostatic potential-modulated spin relaxation magnetic resonance.Science. 1998; 279: 1925-1929Crossref PubMed Scopus (108) Google Scholar). The secreted enzymes show similar activity to phospholipids with different fatty acids in the sn-2 position (22Singer A.G. Ghomashchi F. Calvez C.Le Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. al et Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2.J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). However they have different preferences for the charge on the lipid surface. PLA2s containing a tryptophan in the lipid binding surface display the highest activity toward neutral lipid substrates, and PLA2s with an excess of basic residues on the lipid binding surface display the highest activity toward negatively charged surfaces (22Singer A.G. Ghomashchi F. Calvez C.Le Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. al et Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2.J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). For a more detailed review of the mechanism of binding to differently charged membranes, see Ref. 23Lambeau G. Gelb M.H. Biochemistry and physiology of mammalian secreted phospholipases A2.Annu. Rev. Biochem. 2008; 77: 495-520Crossref PubMed Scopus (417) Google Scholar.The primary role of the mammalian secreted PLA2 enzymes in eicosanoid signaling remains unclear and has been recently reviewed (23Lambeau G. Gelb M.H. Biochemistry and physiology of mammalian secreted phospholipases A2.Annu. Rev. Biochem. 2008; 77: 495-520Crossref PubMed Scopus (417) Google Scholar). The most well-understood function of a mammalian sPLA2 is group IIA, which has been shown to be a potent antimicrobial agent. Many different studies have examined the role the secreted PLA2s play in eicosanoid release, and these studies have been inconclusive. They show that the up-regulation of groups IIA, V, and X caused a cytosolic group IVA (GIVA) PLA2 dependent increase in eicosanoids. However a specific inhibitor of the group IIA inhibitor has been developed by Schevitz et al. (24Schevitz R.W. Bach N.J. Carlson D.G. Chirgadze N.Y. Clawson D.K. Dillard R.D. Draheim S.E. Hartley L.W. Jones N.D. Mihelich E.D. al et Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2.Nat. Struct. Biol. 1995; 2: 458-465Crossref PubMed Scopus (233) Google Scholar), with clinical trials of its efficacy against arthritis and allergens showing no therapeutic effects (23Lambeau G. Gelb M.H. Biochemistry and physiology of mammalian secreted phospholipases A2.Annu. Rev. Biochem. 2008; 77: 495-520Crossref PubMed Scopus (417) Google Scholar). The proinflammatory role of the secreted PLA2 has been suggested to possibly be controlled by a protein binding event not dependent on PLA2 activity. Receptors present in mouse tissues named the M-type receptors have been shown to bind different secreted phospholipases, but no M-type receptor in humans has been found that binds PLA2 (25Lambeau G. Lazdunski M. Receptors for a growing family of secreted phospholipases A2.Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Recent work however has shown that group IIA PLA2 binds to integrins, and this raises the interesting possibility that integrin-PLA2 contacts may mediate proinflammatory activity (26Saegusa J. Akakura N. Wu C.Y. Hoogland C. Ma Z. Lam K.S. Liu F.T. Takada Y.K. Takada Y. Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins {alpha}v{beta}3 and {alpha}4{beta}1 and induces proliferation of monocytic cells in an integrin-dependent manner.J. Biol. Chem. 2008; 283: 26107-26115Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar).CYTOSOLIC PLA2The first group IV cytosolic PLA2, GIVA, was identified in human platelets in 1986 (27Kramer R.M. Checani G.C. Deykin A. Pritzker C.R. Deykin D. Solubilization and properties of Ca2+-dependent human platelet phospholipase A2.Biochim. Biophys. Acta. 1986; 878: 394-403Crossref PubMed Scopus (68) Google Scholar) and was cloned and sequenced in 1991 (7Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 8Kramer R.M. Roberts E.F. Manetta J. Putnam J.E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells.J. Biol. Chem. 1991; 266: 5268-5272Abstract Full Text PDF PubMed Google Scholar). Many different submembers of the group IV family have been discovered since then and their properties are reviewed (28Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Properties of the group IV phospholipase A2 family.Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar). The most well-studied cytosolic enzyme is the GIVA PLA2. It is characterized by an active site serine and aspartic acid dyad, requirement for Ca2+ for activity, and it is the only PLA2 with a preference for arachidonic acid in the sn-2 position of phospholipids (7Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP.Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 28Ghosh M. Tucker D.E. Burchett S.A. Leslie C.C. Properties of the group IV phospholipase A2 family.Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar). GIVA PLA2 also possesses lysophospholipase activity, as well as transacylase activity (29Reynolds L. Hughes L. Louis A.I. Kramer R.A. Dennis E.A. Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2.Biochim. Biophys. Acta. 1993; 1167: 272-280Crossref PubMed Scopus (151) Google Scholar). Arachidonic acid is the precursor for the generation of eicosanoids, and this enzyme has been proposed to play a major role in inflammatory diseases. This was proven through the use of knockout mouse models, where the absence of the GIVA PLA2 gene significantly reduced the effects of many inflammatory diseases (30Bonventre J.V. Huang Z. Taheri M.R. Li E.O'Leary, E. Moskowitz M.A. Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 31Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. al et Role of cytosolic phospholipase A2 in allergic response and parturition.Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar, 32Uozumi N. Shimizu T. Roles for cytosolic phospholipase A2alpha as revealed by gene-targeted mice.Prostaglandins Other Lipid Mediat. 2002; 68–69: 59-69Crossref PubMed Scopus (73) Google Scholar). GIVA PLA2 is now generally considered to be a central enzyme mediating generation of eicosanoids and hence many inflammatory processes.The structure of this enzyme shows that it is composed of a Ca2+ dependent lipid binding C2 domain, and a catalytic α/β hydrolase domain as shown in Fig. 2A(33Dessen A. Tang J. Schmidt H. Stahl M. Clark J.D. Seehra J. Somers W.S. Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism.Cell. 1999; 97: 349-360Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Both of these domains are required for full activity (34Hsu Y.H. Burke J.E. Stephens D.L. Deems R.A. Li S. Asmus K.M. Woods Jr., V.L. Dennis E.A. Calcium binding rigidifies the C2 domain and the intra-domain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.J. Biol. Chem. 2008; 283: 9820-9827Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The catalytic domain of the enzyme is composed of a core α/β hydrolase region conserved throughout many different lipases, as well as a novel cap region found only in GIVA PLA2. Within the cap region, there is a lid region that prevents the modeling of a phospholipid substrate in the active site. It has been proposed that this enzyme must undergo a conformational change in the presence of substrate that opens this lid region. Recent work using lipid substrate, as well as a covalent inhibitor bound in the active site, has indeed shown a conformational change of the lid region in the presence of substrate (35Burke J.E. Hsu Y.H. Deems R.A. Li S. Woods Jr., V.L. Dennis E.A. A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2.J. Biol Chem. 2008; 283: 31227-31236Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).Fig. 2A: Group IVA PLA2 crystal structure as determined by Dessen et al. (33Dessen A. Tang J. Schmidt H. Stahl M. Clark J.D. Seehra J. Somers W.S. Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism.Cell. 1999; 97: 349-360Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The C2 domain is shown in orange, with two bound Ca2+ ions shown in purple. The catalytic domain is shown on the right with the cap region colored yellow, and the lid region 415–432 colored magenta. The active site residues Ser-228, Asp-549, and Arg-200 are shown in stick form colored red. The PIP2 binding site is shown in dark blue, and the C1P binding site is shown in cyan. B: Model of the lipid-binding surface of the group IVA (GIVA) PLA2 with residues colored based on interaction with lipid membrane. Adapted from Burke et al (35Burke J.E. Hsu Y.H. Deems R.A. Li S. Woods Jr., V.L. Dennis E.A. A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2.J. Biol Chem. 2008; 283: 31227-31236Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)This enzyme is activated by many different mechanisms. The enzyme is recruited to the membrane by a Ca2+ dependent translocation of the C2 domain. Recent work has localized the lipid binding surface of the enzyme in the presence of Ca2+, as shown in Fig. 2B (35Burke J.E. Hsu Y.H. Deems R.A. Li S. Woods Jr., V.L. Dennis E.A. A phospholipid substrate molecule residing in the membrane surface mediates opening of the lid region in group IVA cytosolic phospholipase A2.J. Biol Chem. 2008; 283: 31227-31236Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The lipid second messengers ceramide-1-phosphate (36Stahelin R.V. Subramanian P. Vora M. Cho W. Chalfant C.E. Ceramide-1-phosphate binds group IVA cytosolic phospholipase a2 via a novel site in the C2 domain.J. Biol. Chem. 2007; 282: 20467-20474Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and phosphatidylinositol (4Seilhamer J.J. Pruzanski W. Vadas P. Plant S. Miller J.A. Kloss J. Johnson L.K. Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid.J. Biol. Chem. 1989; 264: 5335-5338Abstract Full Text PDF PubMed Google Scholar, 5Kramer R.M. Hession C. Johansen B. Hayes G. McGray P. Chow E.P. Tizard R. Pepinsky R.B. Structure and properties of a human non-pancreatic phospholipase A2.J. Biol. Chem. 1989; 264: 5768-5775Abstract Full Text PDF PubMed Google Scholar) bisphosphate (37Six D.A. Dennis E.A. Essential Ca(2+)-independent role of the group IVA cytosolic phospholipase A(2) C2 domain for interfacial activity.J. Biol. Chem. 2003; 278: 23842-23850Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) have been shown to activate the enzyme and work using site-directed mutagenesis has identified two charged residue patches on the enzyme that bind these lipid second messengers. The enzyme has also been shown to be regulated through phosphorylation on residues 505, 515, and 727 (see Ref. 38Burke J.E. Dennis E.A. Phospholipase A2 biochemistry.Cardiovasc. Drugs Ther. 2008; (In press)PubMed Google Scholar).Recognition of the importance of the GIVA PLA2 in inflammatory diseases, as well as important structural discoveries has made it a very attractive drug target, and many different laboratories have attempted to develop inhibitors. Two of the most promising drug candidates include the indole derivative inhibitors developed by Wyeth, and the 2-oxoamide inhibitors developed by Six et al. (39Six D.A. Barbayianni E. Loukas V. Stephens V.Constantinou-Kokotou, D.Hadjipavlou-Litina, D. Wong A.C. Magrioti V. Baker P.Moutevelis-Minakakis, S.F. al et Structure-activity relationship of 2-oxoamide inhibition of group IVA cytosolic phospholipase A(2) and group v secreted phospholipase A(2).J. Med. Chem. 2007; 50: 4222-4235Crossref PubMed Scopus (70) Google Scholar) and McKew et al. (40McKew J.C. Lee K.L. Shen M.W. Thakker P. Foley M.A. Behnke M.L. Hu B. Sum F.W. Tam S. Hu Y. al et Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(dipheny lmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.J. Med. Chem. 2008; 51: 3388-3413Crossref PubMed Scopus (82) Google Scholar). Both of these inhibitors have been used for in vivo animal models of inflammation and have shown potency in reducing inflammatory effects (40McKew J.C. Lee K.L. Shen M.W. Thakker P. Foley M.A. Behnke M.L. Hu B. Sum F.W. Tam S. Hu Y. al et Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(dipheny lmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.J. Med. Chem. 2008; 51: 3388-3413Crossref PubMed Scopus (82) Google Scholar, 41Yaksh T.L. Kokotos G. Svensson C.I. Stephens D. Kokotos C.G. Fitzsimmons B. Hua D.Hadjipavlou-Litina, X.Y. Dennis E.A. Systemic and intrathecal effects of a novel series of phospholipase A(2) inhibitors on hyperalgesia and spinal prostaglandin E-2 release.J. Pharmacol. Exp. Ther. 2006; 316: 466-475Crossref PubMed Scopus (63) Google Scholar). Potential side effects of GIVA PLA2 inhibitors have been suggested by recent work examining a human patient with defects in GIVA PLA2 who showed decreases in PLA2 activity, eicosanoid biosynthesis, and the generation of many small intestinal ulcers (42Adler D.H. Cogan J.D. Milne J.A. Phillips 3rd, N.Schnetz-Boutaud, G.L. Iverson T. Stein J.A. Brenner D.A. Morrow J.D. Boutaud O. al et Inherited human cPLA(2alpha) deficiency is associated with impaired eicosanoid biosynthesis, small intestinal ulceration, and platelet dysfunction.J. Clin. Invest. 2008; 118: 2121-2131PubMed Google Scholar).Ca2+ INDEPENDENT PLA2The Ca2+ independent PLA2s are members of the GVI family of PLA2 enzymes. The first member of this family the GVIA PLA2 was purified from macrophages in 1994 (12Ackermann E.J. Kempner E.S. Dennis E.A. Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar). All of the GVI enzymes are characterized by not requiring Ca2+ for catalytic activity. Many new GVI PLA2 members have been identified in the last three years (as reviewed in Ref. 14Schaloske R.H. Dennis E.A. The phospholipase A2 superfamily and its group numbering system.Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (720) Google Scholar). The best characterized of the GVI PLA2 enzymes is the GVIA PLA2 (43Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). It is found in cells expressed in multiple different splice variants (44Winstead M.V. Balsinde J. Dennis E.A. Calcium-independent phospholipase A(2): structure and function.Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (219) Google Scholar). The active splice forms of the enzyme GIVA-1, and GIVA-2 are composed of 7-8 ankyrin repeats, a linker region and a catalytic domain. This enzyme, similar to GIV PLA2, uses a serine in the active site to catalyze the cleavage of the sn-2 ester bond; however it does not show specificity for an arachidonic acid in the sn-2 position. The GVIA PLA2 also posseses a lysophospholipase activity, as well as transacylase activity (44Winstead M.V. Balsinde J. Dennis E.A. Calcium-independent phospholipase A(2): structure and function.Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (219) Google Scholar). The activity of the GVIA PLA2 has been suggested to be regulated through many different mechanisms, including ATP binding, caspase cleavage, calmodulin, and possible ankyrin repeat mediated protein aggregation (38Burke J.E. Dennis E.A. Phospholipase A2 biochemistry.Cardiovasc. Drugs Ther. 2008; (In press)PubMed Google Scholar).The role of the GVIA PLA2 in different signaling pathways has been shown to be very complex. Initial reports of the functions of the GVIA PLA2 were determined using the inhibitor bromoenollactone (44Winstead M.V. Balsinde J. Dennis E.A. Calcium-independent phospholipase A(2): structure and function.Biochim. Biophys. Acta. 2000; 1488: 28-39Crossref PubMed Scopus (219) Google Scholar). Recent work has shown that this inhibitor is not specific for GVIA PLA2 and actually functions through activation of the inhibitor by GVIA PLA2 followed by nonspecific covalent modification of cysteine residues in all proximally located enzymes (45Song H. Ramanadham S. Bao S. Hsu F.F. Turk J. A bromoenol lactone suicide substrate inactivates group VIA phospholipase A2 by generating a diffusible bromomethyl keto acid that alkylates cysteine thiols.Biochemistry. 2006; 45: 1061-1073Crossref PubMed Scopus (47) Google Scholar). Therefore it has been hard to evaluate early experiments using this inhibitor to determine the function of the GVIA PLA2. Experiments using the inhibitor bromoenollactone are reviewed elsewhere (46Balsinde J. Balboa M.A. Cellular regulation and proposed biological functions of group VIA calcium-independent phospholipase A(2) in activated cells.Cell. Signal. 2005; 17: 1052-1062Crossref PubMed Scopus (181) Google Scholar). Two major factors have allowed the determination of GVI PLA2’s cellular functions. First the recent generation of GVIA PLA2 deficient mice has shown the importance of this enzyme in bone formation, apoptosis, insulin secretion, and sperm development (47Bao S. Miller D.J. Ma Z. Wohltmann M. Eng G. Ramanadham S. Moley K. Turk J. Male mice that do not express group VIA phospholipase A2 produce spermatozoa with impaired motility and have greatly reduced fertility.J. Biol. Chem. 2004; 279: 38194-38200A
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