This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The intracellular form of platelet activating factor acetylhydrolase (PAFAH-II) functions by reducing levels of the signaling molecule platelet activating factor (PAF) as a general anti-inflammatory scavenger and is linked to asthma, allergic reactions and atherosclerosis. Physiologically, the intracellular form PAFAH-II is believed to be functionally active after association with the inner-leaflet of kidney and liver cells. The goal of this project is to elucidate the mechanism of the oxidative stress response in PAFAH-II. The molecular understanding of structure and function of PAFAH-II will directly impact human health. We will tie together a structural perspective, biophysical characterization, and in vivo localization and oxidative regulation for the PAFAH-II enzyme.
We aim to elucidate the relationship between structure and interfacial function of PAFAH-II in two related ways. 1) PAFAH-II is N-terminally myristoylated in vivo, and this myristoyl motif is believed to direct the enzyme to the inner leaflet of cells following an oxidative stress response.
We aim to characterize the role of the myristoyl tail for membrane localization using both in vitro and in situ approaches. Expression in E. coli with coexpressed myristoyl transfer enzymes will allow a biophysical and biochemical characterization of PAFAH-II binding to PC vesicles. In parallel we will express PAFAH-II as a fusion with cyan fluorescent protein in human kidney cells followed by confocal microscopy localization. This will provide a better understanding of the function of the myristoyl motif on the N-terminus of PAFAH-II. 2) Following the successful expression of myristoylated PAFAH-II in E. coli and human kidney cells we will define the mechanism behind the oxidative stress response that leads to PAFAH-II localization to the cell membrane. Initial experiments will focus on characterizing the result of oxidative stress using the in vitro system of E. coli expressed myristyol and non-myristoyl PAFAH-II. Promising leads will then be explored in the human kidney cells to validate the physiological relevance of our findings.
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