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.
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