Platelet-activating factor acetylhydrolases, PAF-AHs, are ubiquitous enzymes found in the cell cytosol in many tissues as well as in the blood. These enzymes function as off switches for the phospholipid messenger PAF, and inactivate the latter by hydrolysis of the acetyl group in the sn-2 position. PAF is a very potent messenger molecule, known particularly for the cell-activating and proinflammatory functions of the plasma pool, functions that are mediated by G-protein-coupled receptors. The intracellular PAF has been implicated in a wide variety of signaling pathways, where it plays a role of the second messenger. The brain contains three intracellular isoforms of PAF-AHs. PAF-AH(Ib) is a trimer of two homologous catalytic subunits (a1 and a2, 26 kD each) and a regulatory subunit b (beta) (45kD), a member of the WD40 family of proteins. The expression of all three subunits is regulated during fetal and adult lives at the transcription level. The deletion of the gene (LIS-1) coding for the b-subunit causes a developmental brain lissencephaly, in which no cortex is formed due to impaired neuron migration. We have recently shown by high-resolution x-ray crystallography that the catalytic subunits share of common fold with Ras, and that the hydrolytic site is located topologically close to the GTP-ase site in Ras. The structure of the b-subunit, as inferred from sequence alignment, is similar to that of b-subunit of the trimeric G-protein transducin. There is evidence that it signals in the fetal brain to the cytoskeleton through a tight complex with tubulin. PAF-AH(II) is a member of the a/b (alpha/beta) hydrolase superfamily and may act both as a scavenger of inflammatory phospholipids, including PAF, or as an off switch in PAF-mediated signal transduction. The plan is to study the structure-function relationships in both isoforms, to better understand the interactions between the subunits in the trimeric PAF-AH(Ib) and its role in neuronal migration in developing brain. This will be accomplished by extensive crystallographic analyses followed by mutational and functional studies in our laboratory and those of several collaborators. To understand the relationships between the different isoforms of PAF-AHs, the investigators will also study the isoform II, for which an atomic model has been generated using a high-resolution (1.9A) structure of the protein s homolog, a bacterial lipase. Finally, plans include structural studies of the NudF gene product from Aspergillus, nearly 50% identical to LIS-1, and responsible for nuclear translocation in the fungus; the belief is that the movement of nuclei precedes the migration of neurons.
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