The goal of this project is to characterize the molecular properties of Platelet-Activating Factor acetylhydrolase (PAF-AH), an anti- inflammatory phospholipase A2 with high specificity for hydrolysis of PAF and structurally related phospholipids with potent biological activities. PAF is a phospholipid hormone that activates platelets, neutrophils and other inflammatory cells at nanomolar concentrations, through a specific receptor. PAR can cause anaphylaxis, sepsis an death, which suggests that regulatory mechanisms must exist to prevent inappropriate accumulation of this lipid autacoid. The levels of PAR are precisely controlled at key independent steps during synthesis. In addition, PAF-AH activities are ubiquitously present in active form in tissues and blood to insure rapid and efficient PAR degradation at all locations. However, we and other have shown that uncontrolled oxidation of certain phospholipids results in the formation of compounds that can activate the PAR receptor in a manner identical to that of PAR. The pro- inflammatory and thrombotic effects of these compounds can only be controlled at the degradative level because synthesis is dictated by the concentration of oxidants and free radicals produced as by-products of multiple reactions. This proposal focuses on the secreted form of PAR acetylhydrolase which circulates without acting on phosphatidylcholines containing long sn-2 acyl groups. The enzyme has high affinity for PAF and phospholipids containing short or oxidized sn-2 groups produced during oxidation of LDL and membrane phospholipids. PAF-AH circulates as a complex with lipoproteins which the enzyme protects as it scavenges lipids produced as a consequence of oxidative stress. We have cloned the cDNA encoding the plasma form of PAF-AH and identified regulatory mechanisms that control the expression of the PAF-AH gene. We propose to perform functional and structural studies aimed at characterizing the role played by this enzyme in human physiology and disease.
The Specific Aims of the proposed research include: I. Definition of molecular mechanisms involved in the production and modification of intracellular and secreted forms of PAF-AH; II. Definition of the molecular basis for oxidant inactivation of PAF-AH; III. The investigation of the substrate specificity of PAF acetylhydrolase, with emphasis on oxidized substrates; IV. The determination of the molecular basis for the specificity of interaction between PAF-AH and LDL and V. The determination of the crystal structure of the plasma form of PAF-AH.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL035828-16
Application #
6388991
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Ganguly, Pankaj
Project Start
1985-12-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
16
Fiscal Year
2001
Total Cost
$253,164
Indirect Cost
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Ajiro, Yoichi; Saegusa, Noriko; Giles, Wayne R et al. (2011) Platelet-activating factor stimulates sodium-hydrogen exchange in ventricular myocytes. Am J Physiol Heart Circ Physiol 301:H2395-401
Matsumiya, Tomoh; Stafforini, Diana M (2010) Function and regulation of retinoic acid-inducible gene-I. Crit Rev Immunol 30:489-513
Gardner, Alison A; Reichert, Ethan C; Alexander, Timothy S et al. (2010) Novel mechanism for regulation of plasma platelet-activating factor acetylhydrolase expression in mammalian cells. Biochem J 428:269-79
Lu, Jing; Pierce, Marissa; Franklin, Andrew et al. (2010) Dual roles of endogenous platelet-activating factor acetylhydrolase in a murine model of necrotizing enterocolitis. Pediatr Res 68:225-30
Edelstein, Celina; Pfaffinger, Ditta; Reichert, Ethan C et al. (2010) Mouse plasminogen has oxidized phosphatidylcholine adducts that are not metabolized by lipoprotein-associated phospholipase A?under basal conditions. Int J Mol Sci 11:5339-47
McIntyre, Thomas M; Prescott, Stephen M; Stafforini, Diana M (2009) The emerging roles of PAF acetylhydrolase. J Lipid Res 50 Suppl:S255-9
Stafforini, Diana M (2009) Functional Consequences of Mutations and Polymorphisms in the Coding Region of the PAF Acetylhydrolase (PAF-AH) Gene. Pharmaceuticals (Basel) 2:94-117
Stafforini, Diana M (2009) Biology of platelet-activating factor acetylhydrolase (PAF-AH, lipoprotein associated phospholipase A2). Cardiovasc Drugs Ther 23:73-83
Foulks, Jason M; Marathe, Gopal K; Michetti, Noemi et al. (2009) PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 113:6699-706
Gardner, Alison A; Reichert, Ethan C; Topham, Matthew K et al. (2008) Identification of a domain that mediates association of platelet-activating factor acetylhydrolase with high density lipoprotein. J Biol Chem 283:17099-106

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