Lipid metabolism has taken the stage front and center as a human health concern. Here much attention has been focused on the mechanisms involved in inflammation and lipid homeostasis of lipids linked to low density lipoprotein (LDL) and high density lipoprotein (HDL). Many proteins that are associated with these LDL and HDL particles have emerged as playing a critical role in these lipid pathways. The plasma form of platelet activating factor acetylhydrolase (pPAFAH) functions on the surface of LDL particles by reducing levels of the signaling molecule platelet activating factor (PAF) as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma and allergic reactions. A homologous intracellular form, referred to as PAFAH-II, is believed to have similar functions in liver and kidney cells. The phospholipid-associated pPAFAH and PAFAH-II enzymes are worthy structural targets. Physiologically, these enzymes are found associated with LDL particles or the inner leaflet of cells, and as such, are considered interfacial enzymes, which function on the lipid-aqueous interface. In addition to a role to reduce PAF levels, they have been implicated in hydrolytic activities of other pro-inflammatory agents, such as oxidized lipids. We will elucidate the relationship between structure and interfacial function for PAF AH via 4 aims: (i) The heterologous expression of and use of additives/detergents will be screened in order to obtain homogeneous forms of the pPAFAH and PAFAH-II enzymes. The quality of protein will be assessed by biophysical characterization, functional assay and protein crystal growth in order to obtain monodisperse and soluble forms of PAFAH suitable for structural and functional studies, (ii) The high-resolution crystal structure of the phospholipid- associated PAFAH enzymes will be solved. The use of detergents and amphiphilic molecules will be explored to provide higher resolution structures, as well as functionally more relevant structures, (iii) Inhibitors and substrate-mimics of PAFAH will be explored and developed via structural and kinetic characterization to elucidate in vivo physiological functions, (iv) The physiologically relevant reaction of PAFAH with organophosphate (OP) compounds will be characterized. We are interested in obtaining structural models to develop the LDL-associated pPAFAH as a practical therapeutic for people exposed to these toxic organophosphate insecticides and nerve agents.
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