INVESTIGATION INTO THE REDOX-SENSITIVE, OLIGOMERIC STATE-BASED REGULATION OF THE ANTIOXIDANT ENZYME PLATELET ACTIVATING FACTOR ACETYLHYDROLASE TYPE II

Abstract

Platelet activating factor acetylhydrolase type II (PAFAH2) is an N-terminally myristoylated phospholipase that hydrolyzes oxidized phospholipids in intracellular membranes. It has been implicated in cardiovascular disease due to this activity. PAFAH2 has been shown to migrate to membranes from the cytosol in a redox dependent manner and it resolves as two distinct bands during native PAGE. It is believed that PAFAH2’s cellular trafficking and activity is regulated by its oligomeric state and that an oxidative post translational modification drives the dissociation of the water-soluble dimer to the membrane associating monomer. Two expression systems for the generation of wildtype and a fluorescent fusion of PAFAH2 were developed. PAFAH2 was expressed in SF21 insect cells. SF21 expressed PAFAH2 resolved as two, higher than expected bands on SDS PAGE. LC-MS/MS confirmed these bands to both be PAFAH2 with the higher molecular weight band having a coverage of 65.05% and the lower band of 97.70%. The higher coverage band was not detected as being N-terminally myristoylated. A total of seven potential phosphorylation sites were identified for the higher coverage band. SF21 expressed wildtype PAFAH2 demonstrated 92 times and 13 times the activity for PNPB of uninfected cells and the fluorescent PAFAH2 fusion, respectively. Attempts to co-express wildtype PAFAH2 in E. coli with NMT were made but results were inconclusive. GROMACS was used to conduct MD simulations with wildtype and oxidatively modified PAFAH2 dimers. Sulfenylation of C72 produced lower average RMSD and RMSDist compared to wildtype PAFAH2. Hydrogen bonding between sulfenylated C72 and the unmodified C72 of the adjacent subunit of the dimer appeared to have a stabilizing effect, strengthening the interactions at the dimer interface.