Characterization of human platelet activating factor acetylhydrolase type-II oxidative stress response

Platelet-activating factor acetylhydrolase type-II (PAFAH-II) is an intracellular enzyme with the ability to cleave oxidatively fragmented phospholipids. It is found in a variety of cell types, simultaneously in the cytoplasm and bound to the inner membranes. Introduction of oxidative stress prompts PAFAH-II to redistribute itself to be more membrane bound. Numerous studies have shown PAFAH-II protects cells from oxidative stress induced apoptosis, presumably by cleaving the oxidatively fragmented portion of a damaged phospholipid. By trafficking from the cytoplasm to the membrane, PAFAH-II can be localized to the areas containing damaged phospholipids. However, studies have not gone so far as to include membrane binding structural studies or exact cellular location of this enzyme. Additionally, the stress response mechanism of PAFAH-II remains unresolved and a crystal structure has yet to be determined.;The work described here begins to examine the oxidative stress response of PAFAH-II and aims to gain a better understanding of the physiological role of this enzyme. Using the PAFAH-II homology model as a guide, experiments to determine the structural requirements for membrane binding and oligomerization of PAFAH-II were completed. Recombinant expression and confocal imagining of various PAFAH-II constructs in HEK293 cells showed that the myristoyl group and five hydrophobic residues are required for enzyme localization to the membrane. Co-localization experiments with organelle specific labels determined that PAFAH-II specifically localizes to the membranes of Golgi and Endoplasmic Reticulum.;Native PAGE and photon counting histogram showed that PAFAH-II forms oligomers, and is specifically a dimer in the cytoplasm and a monomer when bound to the membrane. The myristoyl group and hydrophobic patch are also shown to play a role in enzyme dimerization. The work presented here also describes purification of enzymatically stable PAFAH-II from E. coli and S. cerevisiae. Emphasis is placed on producing myristoylated PAFAH-II, to be used in future structural and in vitro work.;We propose that the oxidative stress response of PAFAH-II is regulated by the oligomeric state of the protein. In unstressed conditions, PAFAH-II is a cytoplasmic dimer, oriented in a head to tail fashion. When oxidative stress is introduced, the PAFAH-II dimer dissociates into monomers. The PAFAH-II monomer traffics to the membranes of the Golgi and Endoplasmic Reticulum, binding to the bilayer by the myristoyl group and hydrophobic patch residues.