Deciphering the Interaction of FLAP and 5LO

Abstract

Leukotrienes are pro-inflammatory lipid mediators involved in chronic inflammatory diseases like asthma and atherosclerosis. Different enzymes and proteins are involved in the leukotriene biosynthesis pathway that stems from oxygenation of arachidonic acid (AA) by 5LO (5-lipoxygenase). FLAP (5-lipoxygenase activating protein) is an integral membrane protein, belonging to the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism) family. Till now, there is no prominent evidence for any biochemical or enzymatic activity for FLAP, except involvement as an “activator” in leukotriene biosynthesis. The hypothesis is that, on increase in intracellular calcium concentration, 5LO moves from the cytosol to the nuclear membrane and localizes near FLAP. The AA is then transferred from the nuclear membrane to 5LO by homo-trimeric FLAP. Then 5LO converts the AA to leukotriene A4. This interaction between FLAP and 5LO is ambiguous due to intricate mechanisms which occur at the interface of the nuclear membrane and also between an enzyme and an integral membrane protein with no documented function. To understand the function and involvement of FLAP in leukotriene biosynthesis in vitro, we employ soluble phospholipid bilayers called “Nanodiscs” which mimic a membrane environment. The nanodisc will act as a stable platform for structural and functional characterization of the interaction between 5LO and FLAP. After initial studies of the Ca2+dependent recruitment of 5LO to empty nanodiscs as well as the reconstitution of FLAP into nanodiscs, the entire complex 5LO-FLAP-nanodisc can be targeted. We employ biochemical assays to characterize the interactions and transmission electron microscopy (single particle analysis) to create a 3D model of the functional complex of 5LO and FLAP. Here, we portray our outcomes from the above mentioned subprojects encompassed in understanding the interaction of 5LO and FLAP.