ELUCIDATION OF 15-LIPOXYGENASE-2 AND PEBP1 INTERACTIONS IMPLICATED IN ACUTE RENAL FAILURE

Abstract

15-Lipoxygenase-2 (15-LOX-2) is one of six human lipoxygenase enzymes that catalyze the peroxidation of fatty acids and are involved in many different cell signaling pathways related to development, homeostasis, and even disease. 15-LOX-2 enzyme has recently been implicated in the ferroptosis pathway, or iron dependent programmed cell death, that is linked to acute renal failure. It is hypothesized that this occurs when phosphatidylethanolamine binding protein 1 (PEBP1) associates with 15-LOX-2 at the cellular membrane, leading to an allosteric change 15-LOX-2’s structure. This interaction has been proposed to alter 15-LOX-2’s substrate affinity from free fatty acids to phospholipids (primarily derived from phosphatidylethanolamine, PE, acquired from the membrane), resulting in the generation of hp-ETE-PE’s that feed into ferroptosis pathways when they are not adequately reduced by glutathione peroxidase IV complex. Despite this emerging model, there is no biochemical evidence for the PEBP1/15-LOX-2 interaction or allosteric regulation. Resolving this interaction is important towards developing methodologies for small molecule intervention approaches, such as employing recently discovered selective inhibitor of 15-LOX-2. For this thesis, interactions between 15-LOX-2, PEBP1, and the cell membrane are investigated using 10 nm lipid nanodiscs as a model of the phospholipid bilayer. Fast protein liquid chromatography (FPLC) and SDS-PAGE show binding of the 15-LOX-2 to the first generation nanodisc prep.