Elucidating the Interaction of 5-Lipoxygenase and FLAP

Abstract

Inflammation is one of the innate defense mechanisms exerted by the human body for protection and to initiate the healing process. Prolonged inflammatory reactions can lead to chronic disease conditions like atherosclerosis, asthma and myocardial infarction. Leukotrienes (LTs) are one of several pro-inflammatory lipid mediators involved in such inflammatory diseases and are derived from arachidonic acid (AA). The key enzyme involved in LT biosynthesis is 5 Lipoxygenase (5LO), Five lipoxygenase activating protein (FLAP) an integral membrane protein and Coactosin like protein (CLP) a scaffolding protein. Upon external stimuli, intracellular calcium concentration increases which translocates 5LO from the cytosol to the nuclear membrane and localizes near FLAP. Then 5LO converts the AA to leukotriene A4. The hypothesis is that AA is transferred from the nuclear membrane to 5LO by homo-trimeric FLAP and CLP is also involved with 5LO in this stage. Though this hypothesis has been studied extensively, the association between these proteins in LT biosynthesis is still clouded. To elucidate these assisted interactions, we reconstituted the FLAP into “Nanodisc” a membrane mimicking system. We grouped the project by first analyzing the interaction of 5LO with ND, to simulate and understand the calcium mediated translocation of 5LO to nuclear membrane in this ND system. We then proceeded to use FLAP-containing nanodiscs (FND) and repeated aforementioned analyses. We employed biochemical assays and transmission electron microscopy to characterize the interactions and to create a 3D model of the functional complex of 5LO,CLP and FLAP. Here, we show our results from the above mentioned projects involved in understanding the interaction of proteins involved in the initiation of leukotriene biosynthesis.