Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation/Thesis

Abstract

Leukotrienes (LT) are lipid mediators of the inflammatory response that play important roles in diseases such as asthma and atherosclerosis. Leukotriene A4 (LTA4) is synthesized from arachidonic acid (AA) by 5-lipoxygenase (5-LOX) with the help of 5-lipoxygenase activating protein (FLAP), a trimeric nuclear transmembrane protein. Exactly how 5-LOX and FLAP interact is not well understood, however FLAP is essential for the production of leukotrienes in vivo. I used nanodiscs (NDs), engineered membrane systems, as a tool to study the 5-LOX-FLAP interaction. FLAP was incorporated into NDs with a phospholipid composition to mimic of the nuclear membrane. Structural features were observed with single-particle negative stain transmission electron microscopy (TEM). This is the first in vitro reconstitution of this protein-protein-membrane interaction.

I demonstrated that a distinct 5-LOX sequence motif (tri-lysine motif) modulates activity and confers dependence on FLAP. Substitution of motif with a highly conserved sequence found in homologous enzymes results in ~20-fold higher 5-LOX product levels in stimulated HEK cells relative to the wild-type-expressing cells. However, despite the fact that the presence of FLAP increases product levels in wild-type-expressing cells, it significantly reduces the amount of product detected in cells which express the mutant enzyme. This data suggests that the tri-lysine motif limits enzyme activity and that this inhibition must be compensated by the presence of FLAP for effective LT biosynthesis.

5-LOX activity is modulated by natural products. The frankincense derived compound 3-Acetyl-11-Keto-beta Boswellic Acid (AKBA) is a well-known inhibitor of 5-LOX. We investigated the mechanism of inhibition of 5-LOX by AKBA and defined a novel allosteric binding site. In the presence of FLAP, AKBA effectively blocks the initiation of LT production in HEK cells. Data from immuno-fluorescence microscopy and in vitro studies suggest that AKBA interferes with the 5-LOX:FLAP interaction. Combined, these studies support a specific 5-LOX-FLAP-membrane interaction and provide a path to define the molecular details of this interaction.

Date

3-19-2018

Committee Chair

Newcomer, Marcia

Available for download on Tuesday, March 19, 2019

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