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Research Activities

Overview

    
The Dennis group has studied the structure and function of phospholipases for the past 28 years. We have extensively characterized the cobra venom phospholipase A2 and from these studies have developed a model for the action of soluble enzymes at phospholipid surfaces. These studies have been extended to numerous other phospholipases. We have also explored the role that these enzymes play in various cell function in macrophages, aminiotic WISH cells, and in neural tissues.
Since phospholipase A2 liberates arachidonic acid, it occupies an important position in the eicosanoid cascade and is partially responsible for the regulation of prostaglandin production. The regulation of the production of these compounds has been shown to be important in inflammation, the onset of premature labor, and the etiology of various neural degenerative diseases.
Thus understanding how phospholipase A2 functions and how to control its activity, offer important avenues for developing potent pharmalogical agents for controlling numerous diseases.   
     

Lipid Second Messengers and Phospholipases
Numerous signal transduction processes involve lipids as signaling molecules. Many of these molecules are generated by phospholipases such as phospholipase A2 (PLA2) which releases fatty acids such as arachidonic acid and lysophospholipids. Each of these products is implicated in signal transduction processes itself, but also serves as a precursor for eicosanoids including the prostaglandins, leukotrienes, and lipoxins or platelet activating factor (PAF). These compounds are implicated in numerous inflammatory diseases such as rheumatoid arthritis, sepsis, intestinal bowel disease (IBD), asthma as well as playing a role in cancer, atherosclerosis and premature parturition.
Other important phospholipases include phospholipase C which controls the production of inositol-1,4,5-trisphosphate (IP3) which induces cytosolic Ca2+ release and diacylglycerol (DAG) which activates protein kinase C. Phospholipase D generates phosphatidic acid (PA) which subsequently can be either metabolized by PLA2 generating lysophosphophatidic acid (Lyso PA), a potent cellular mitogen, or by phosphatidate phosphohydrolase (PAP) yielding DAG. Sphingomyelinase, a phospholipase C type enzyme, and related enzymes of sphingolipid metabolism are implicated in apoptosis and other signaling processes. In summary, the phospholipases generate numerous lipid products which control much of cellular signaling and our aim is to better understand their regulation.

     

Characterization of Phospholipase A2

Our goal is to identify and characterize in vitro all of the individual phospholipases involved in a given cell’s regulation and to also characterize that regulation in the intact cell and tissue. To that end, our laboratory has focused initially on phospholipase A2 [Review: 1] where we obtained three distinct types of PLA in pure form and studied their in vitro activities. This includes several Ca2+-dependent secretory enzymes (sPLA2), the Ca2+-dependent cytosolic enzyme (cPLA2) and a Ca2+-independent PLA2 (iPLA2) [Review: 2] which we were the first to identify, purify and characterize (3) and study its inhibition (4). This Group VI enzyme has been cloned (5). We were the first to find that the Group V sPLA2 is expressed and secreted in response to stimuli (6) and we have identified critical residues in the Group IV cPLA2 (7) and have shown that it contains a binding site for phosphatidylinositol 4,5-bisphosphate (PIP2) which activates the enzyme (8 + 43).

The three enzymes mentioned are the main examples of what is a growing superfamily of phospholipase A2's [Review: 9]. For the in vitro study of these enzymes, we have cloned and expressed human and murine examples of each type of PLA2 using yeast Pichia pastoris, bacterial E. coli and the Baculovirus/Sf9 cell based expression systems and even chemical synthesis (10). We are currently carrying out expression studies, site-directed mutagenesis, kinetic analysis, and NMR and mass spectrometric studies of substrate and inhibitor interactions.
 

Inhibitors and Cellular Studies

The inhibitor studies provide the knowledge and tools to study the novel enzymes in intact cells [Review: 11]. We have developed assays to distinguish each of these three types of enzymes in cell extracts. Our laboratory was the first to develop antisense oligonucleotides to block sPLA2 (12) in intact cells and we have used this technology to distinguish between the PLA2’s present in cells (13) and to inhibit the iPLA2 (14). More importantly, we have designed and developed numerous chemical inhibitors of the phospholipases and studied their mechanism of inhibition (15). We recently designed a new and novel class of cPLA2 inhibitors, the 2-oxo amides (16) and are exploring their effects in vitro, ex vivo, and in vivo animal models of hyperalgesia and pain. These inhibitors have been and will continue to be developed both in vitro and in cellular studies where the function of each PLA2 subtype is to be assessed. We have been able to differentiate the role of each of these enzymes and have developed a model for signal transduction in macrophages where cellular activation leads to the translocation of the cPLA2 from the cytosol to membranes and release of arachidonic acid followed by secretion of sPLA2 outside the cell where arachidonic acid is released and rapidly taken up by the cells to be converted to prostaglandins (17).
We discovered cross-talk between not only the sPLA2 and cPLA2 but also a coupling to one specific cyclooxygenase, COX-2 (18). Unsaturated fatty acids such as arachidonic acid when ingested or when added to cells are rapidly taken up and incorporated into phospholipids. We have shown that the iPLA2 plays a central role in membrane remodeling generating the lysophospholipid precursor that combines with the unsaturated fatty acid (19). This essential enzyme is necessary to provide the storage form of unsaturated fatty acids in phospholipids for release upon signal transduction.
We have more recently expanded our understanding of the novel regulatory cascade we discovered for cPLA2/sPLA2/COX-2 which is induced by LPS (20,21,22) and have now identified a number of distinct activation systems for macrophages including purinergic (23), zymosan (24) and UV light (25). The latter provides a Ca2+-independent mode of activating cPLA2 which may involve PIP2. We are now determining the intracellular localization of these enzymes using confocal microscopy and enhancing our understanding of the subtle levels of PLA2 regulation in macrophages.
 

 Membrane Phenomenon and Surface Dilution Kinetics
 One of the major difficulties in studying enzymes acting in and on membranes and receptors localized therein is that biological phenomenon occur in two dimensional space rather than in the three dimensional solutions that have been traditionally studied by biochemists. In the early 1970’s, we developed a novel approach to such studies called “surface dilution” kinetics [Review: 26] which provides an overall framework for examining the action of a variety of signal transduction enzymes on surfaces. In this system the substrate concentration is expressed in surface dilution units of mole fraction and allows analysis of kinetics. We have also studied these enzymes structurally by x-ray (27) and NMR (28) where we have also examined the interaction with interfaces such as micelles. We are now applying the surface dilution phenomenon to the analysis of activators (29) and inhibitors which in general partition with the surface and must be considered in surface dilution units. This analysis is central to analyzing the activation and inhibition of signal transduction enzymes with multiple binding sites.
 

 Other Novel Enzymes
Our research program has focused mainly on phospholipase A2 and macrophages as a prototype signal transduction system. We are also carrying out studies on phospholipase D (30) and lysophospholipases (31) and have cloned and characterized a novel mammalian lysophospholipase (32). We have also studied the regiospecificity of the important lysophospholipase activity of the Group IV cPLA2 (33). Additionally, we discovered a phosphatidate phosphohydrolase in macrophages (34) and a novel signaling molecule, diacylglycerol pyrophosphate (35), which it breaks down. We have also examined signal transduction in human amnionic cells with an aim to understand the role of PLA2 in parturition (36,37). Of special importance, we are currently identifying additional novel phospholipases and their role in signal transduction including those effecting sphingomyelin synthesis (38). One area of new work is to look at the role of PLA2s involved in Alzheimer’s disease and ischemia.
 

Oxidized Lipids:
The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. One of the oxidative processes involves breakdown and modification of the intrinsic lipids and protein and leads to macrophage recognition and uptake of the oxidized LDL (OxLDL). We have chemically synthesized and fully characterized several unique oxidized phospholipids (OxPL). Synthetic adducts of oxidized phospholipids effectively blocked the binding and uptake of OxLDL by mouse peritoneal macrophages (39) and inhibited phagocytosis of apopotic cells by macrophages (40). Furthermore, they blocked the uptake of OxLDL by cells transfected with CD36 (41). Oxidized fatty acids are found predominantly as components of phospholipids, which can be released by phospholipase A2 (PLA2). We have now initiated studies aimed at identifying the PLA2's involved in their hydrolysis and the role of OxPL in generating epitopes on OxLDL for recognition of macrophage scavenger receptors, and we have recently discovered that Schiff base formation and aldol condensates are key to oxidized lipid effects (42).
 

Significant References

  1. Dennis, E.A., Diversity of Group Types, Regulation, and Function of Phospholipase A2, J.Biol.Chem., 269, 13057-13060 (1994).
  2. Balsinde, J. and Dennis, E.A., Function and Inhibition of Intracellular Calcium-Independent Phospholipase A2, J.Biol.Chem., 272, 16069-16072 (1997).
  3. Ackermann, E.J., Kempner, E.S., and Dennis, E.A., Ca2+-Independent Cytosolic Phospholipase A2 from Macrophage-Like P388D1 Cells. Isolation and Characterization, J.Biol.Chem., 269, 9227-9233 (1994).
  4. Ackermann, E.J., Conde-Frieboes, K., and Dennis, E.A., Inhibition of Macrophage Ca2+-Independent Phospholipase A2 by Bromoenol Lactone and Trifluoromethyl Ketones, J.Biol.Chem., 270 , 445-450 (1995).
  5. Balboa, M.A., Balsinde, J., Jones, S.S., and Dennis, E.A., Identity Between the Ca2+-Independent Phospholipase A2 Enzymes from P388D1 Macrophages and CHO Cells, J.Biol.Chem., 272, 8576-8580 (1997).
  6. Balboa, M.A., Winstead, M.V., Balsinde, J., Tischfield, J.A., and Dennis, E.A., Novel Group V Phospholipase A2 Involved in Arachidonic Acid Mobilization in Murine P388D1 Macrophages, J.Biol.Chem., 271, 32381-32384 (1996).
  7. Pickard, R.T., Chiou, X.G., Strifler, B.A., DeFelippis, M.R., Hyslop, P.A., Tebbe, A.L., Yee, Y.K., Reynolds, L.J., Dennis, E.A., Kramer, R.M., and Sharp, J.D., Identification of essential residues for the catalytic function of 85-kDa cytosolic phospholipase A2. Probing the role of histidine, aspartic acid, cysteine, and arginine, J.Biol.Chem., 271, 19225-19231 (1996).
  8. Mosior, M., Six, D.A., and Dennis, E.A., Group IV Cytosolic Phospholipase A2 Binds with High Affinity and Specificity to Phosphatidylinositol 4,5-Bisphosphate Resulting in Dramatic Increases in Activity, J.Biol.Chem., 273, 2184-2191 (1998).
  9. Six,D.A. and Dennis,E.A., The Expanding Superfamily of Phospholipase A2 Enzymes: Classification and characterization, Biochim.Biophys.Acta, 1488, 1-19 (2000).
  10. Canne,L., Botti,P., Simon,R., Chen,Y., Dennis,E.A., and Kent,S., Chemical Protein Synthesis by Solid Phase Ligation of Unprotected Peptide Segments, J.Am.Chem.Soc., 121, 8720-8727 (1999).
  11. Balsinde,J., Balboa,M.A., Insel,P.A., and Dennis,E.A., Regulation and Inhibition of Phospholipase A2., Ann.Reviews of Pharmacology and Toxicology, 39, 175-189 (1999).
  12. Barbour, S. and Dennis, E.A., Antisense Inhibition of Group II Phospholipase A2 Expression Blocks the Production of Prostaglandin E2 by P388D1 Cells, J.Biol.Chem., 268, 21875-21882 (1993).
  13. Balsinde, J., Barbour, S.E., Bianco, I.D., and Dennis, E.A., Arachidonic Acid Mobilization in P388D1 Macrophages is Controlled by Two Distinct Ca2+-Dependent Phospholipase A2 Enzymes, Proc.Natl.Acad.Sci.U.S.A., 91, 11060-11064 (1994).
  14. Balsinde, J., Balboa, M.A., and Dennis, E.A., Antisense Inhibition of Group VI Ca2+-Independent Phospholipase A2 Blocks Phospholipid Fatty Acid Remodelling in Murine P388D1 Macrophages, J.Biol.Chem., 272, 29317-29321 (1997).
  15. Conde-Frieboes, K., Reynolds, L.J., Lio, Y., Hale, M., Wasserman, H.H., and Dennis, E.A., Activated Ketones as Inhibitors of Intracellular Ca2+-Dependent and Ca2+-Independent Phospholipase A2, J.Am.Chem.Soc., 118, 5519-5525 (1996).
  16. Kokotos,G., Kotsovolou,S., Six,D.A., Constantinou-Kokotou,V., Beltzner,C.C., and Dennis,E.A., Novel 2-Oxo Amide Inhibitors of Human Group IVA Phospholipase A2 , J.Med.Chem, 45, 2891-2893 (2002).
  17. Balsinde, J. and Dennis, E.A., Distinct Roles in Signal Transduction for Each of the Phospholipase A2 Enzymes Present in P388D1 Macrophages, J.Biol.Chem., 271, 6758-6765 (1996).
  18. Balsinde, J., Balboa, M.A., and Dennis, E.A., Functional Coupling Between Secretory Phospholipase A2 and Cyclooxygenase-2 and Its Regulation by Cytosolic Group IV Phospholipase A2, Proc.Natl.Acad.Sci.U.S.A., 95, 7951-7956 (1998).
  19. Balsinde, J., Bianco, I.D., Ackermann, E.J., Conde-Frieboes, K., and Dennis, E.A., Inhibition of Calcium-Independent Phospholipase A2 Prevents Arachidonic Acid Incorporation and Phospholipid Remodeling in P388D1 Macrophages., Proc.Natl.Acad.Sci.U.S.A., 92, 8527-8531 (1995).
  20. Shinohara,H., Balboa,M.A., Johnson,C.A., Balsinde,J., and Dennis,E.A., Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s, J.Biol.Chem., 274, 12263-12268 (1999).
  21. Shinohara,H., Lefkowitz,L.J., Johnson,C.A., Balboa,M.A., and Dennis,E.A., Group V Phospholipase A2 Dependent Induction of Cyclooxygenase-2 in Macrophages, J.Biol.Chem., 274, 25967-25970 (1999).
  22. Balsinde,J., Balboa,M.A., Yedgar,S., and Dennis,E.A., Group V Phospholipase A2-Mediated Oleic Acid Mobilization in Lipopolysaccharide-Stimulated P388D1 Macrophages, J.Biol.Chem., 275, 4783-4786 (2000).
  23. Balboa,M.A., Balsinde,J., Johnson,C.A., and Dennis,E.A., Regulation of Arachidonic Acid Mobilization in Lipopolysaccharide-activated P388D1 Macrophages by Adenosine Triphosphate, J.Biol.Chem., 274, 36764-36768 (1999).
  24. Balsinde,J., Balboa,M.A., and Dennis,E.A., Identification of a Third Pathway for Arachidonic Acid Mobilization and Prostaglandin Production in Activated P388D1 Cells, J.Biol.Chem., 275, 22544-22549 (2000).
  25. Balsinde,J., Balboa,M.A., Li,W.-H. , Llopis,L., and Dennis,E.A., Cellular Regulation of Cytosolic Group IV Phospholipase A2 by Phosphatidylinositol Bisphosphate Levels, J.Immunology, 164, 5398-5402 (2000).
  26. Carman, G.M., Deems, R.A., and Dennis, E.A., Lipid-Dependent Enzymes and Surface Dilution Kinetics, J.Biol.Chem., 270, 18711-18714 (1995).
  27. Segelke, B.W., Nguyen, D., Chee, R., Xuong, N.H., and Dennis, E.A., Structures of Two Novel Crystal Forms of Naja naja naja Phospholipase A2 Lacking Ca2+ Reveal Trimeric Packing, J.Mol.Biol., 279, 223-232 (1998).
  28. Plesniak, L., Yu, L., and Dennis, E.A., Conformation of Micellar Phospholipid Bound to the Active Site of Phospholipase A2, Biochemistry, 34, 4943-4951 (1995).
  29. Lefkowitz,L.J., Deems,R.A., and Dennis,E.A., Expression of Group IA Phospholipase A2 in Pichia pastoris: Identification of a Phosphatidylcholine Activator Site Using Site-Directed Mutagenesis, Biochemistry, 38 (43), 14174-14184 (1999).
  30. Balboa, M.A., Balsinde, J., Dennis, E.A., and Insel, P.A., A Phospholipase D-Mediated Pathway for Generating Diacylglycerol in Nuclei from Madin-Darby Canine Kidney Cells, J.Biol.Chem., 270, 11738-11740 (1995).
  31. Wang, A., Deems, R.A., and Dennis, E.A., Cloning, Expression and Catalytic Mechanism of Murine Lysophospholipase I, J.Biol.Chem., 272, 12723-12729 (1997).
  32. Wang, A., Loo, R.W., Chen, Z., and Dennis, E.A., Regiospecificity and Catalytic Triad of Lysophospholipase I, J.Biol.Chem., 272, 22030-22036 (1997).
  33. Loo, R.W., Conde-Frieboes, K., Reynolds, L.J., and Dennis, E.A., Activation, Inhibition, and Regiospecificity of the Lysophospholipase Activity of the 85 kDa Cytosolic Group IV Phospholipase A2, J.Biol.Chem., 272, 19214-19219 (1997).
  34. Balsinde, J. and Dennis, E.A., Bromoenol Lactone Inhibits Magnesium-dependent Phosphatidate Phosphohydrolase and Blocks Triacylglycerol Biosynthesis in Mouse P388D1 Macrophages, J.Biol.Chem., 271, 31937-31941 (1996).
  35. Balboa,M.A., Balsinde,J., Dillon,D.A., Carman,G.M., and Dennis,E.A., Proinflammatory Macrophage-Activating Properties of the Novel Phospholipid Diacylglycerol Pyrophosphate, J.Biol.Chem., 274, 522-526 (1999).
  36. Balboa, M.A., Balsinde, J., and Dennis, E.A., Involvement of Phosphatidate Phosphohydrolase in Arachidonic Acid Mobilization in Human Amnionic WISH Cells, J.Biol.Chem., 273, 7684-7690 (1998).
  37. Johnson,C.A., Balboa,M.A., Balsinde,J., and Dennis,E.A., Regulation of Cyclooxygenase-2 Expression by Phosphatidate Phosphohydrolase in Human Amnionic WISH Cells, J.Biol.Chem., 274, 27689-27693 (1999).
  38. Balsinde, J., Balboa, M.A., and Dennis, E.A., Inflammatory Activation of Arachidonic Acid Signaling via Sphingomyelin Synthase, J.Biol.Chem., 272, 20373-20377 (1997).
  39. Bird,D.A., Gillotte,K.L., Hörkkö,S., Friedman,P., Dennis,E.A., Witztum,J.L., Strifler,B.A., and Steinberg,D., Receptors for Oxidized Low-Density Lipoprotein on Elicited Mouse Peritoneal Macrophages Can Recognize Both the Modified Lipid Moieties and the Modified Protein Moieties: Implications with Respect to Macrophage Recognition of Apoptotic Cells, Proc.Natl.Acad.Sci.U.S.A., 96, 6347-6352 (1999).
  40. Chang,M., Bergmark,C., Laurila,A., Hörkkö,S., Han,K.-H., Friedman,P., Dennis,E.A., and Witztum,J.L., Monoclonal Antibodies Against Oxidized Low-Density Lipoprotein Bind to Apoptotic Cells and Their Phagocytosis by Elicited Macrophages: Evidence that Oxidation-Specific Epitopes Mediate Macrophage Recognition, Proc.Natl.Acad.Sci.U.S.A. , 96, 6353-6358 (1999).
  41. Boullier,A., Gillotte,K.L., Horkko,S., Green,S.R., Friedman,P., Dennis,E.A., Witztum,J.L., Steinberg,D., and Quehenberger,O., The Binding of Oxidized LDL to Mouse CD36 Is Mediated in Part by Oxidized Phospholipids That Are Associated with both the Lipid and Protein Moieties of the Lipoprotein, J.Biol.Chem., 275, 9163-9169 (2000).
  42. Friedman,P., Horkko,S., Steinberg,D., Witztum,J.L., and Dennis,E.A., Correlation of Antiphospholipid Antibody Recognition with the Structure of Synthetic Oxidized Phospholipids: Importance of Schiff Base Formation and Aldol Condensation, J.Biol.Chem., 277, 7010-7020 (2002).
  43. Six, D.A., Dennis, E.A., Essential Ca2+-independent role of the group IV A cytosolic phospholipase A2 C2 domain for interfacial activity, J.Biol.Chem., ( Apr 2 2003, epub ahead of print).
 
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