The role of 5-LOX-activating protein on lipid mediator biosynthesis and its regulation upon bacterial infection

• Applicant: Professorin Ulrike Garscha, Ph.D.
• Subject Area: Pharmacy
• Term: from 2017 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 349867953

Leukotrienes (LT) are pro-inflammatory mediators involved in chronic inflammatory diseases but also in Alzheimer, cancer and cardiovascular diseases. Therefore, pharmacological intervention with LT biosynthesis is a promising therapeutic strategy. The first committed step in LT biosynthesis is the formation of LTA4 from arachidonic acid (AA) by 5-lipoxygenase (5-LOX). Upon stimulation, 5-LOX is activated by diverse mechanisms and translocates to the nuclear membrane where the integral 5-LOX-activating protein (FLAP) resides. Knock-out studies and pharmacological inhibition of FLAP completely abolishes LT biosynthesis in intact leukocytes. Therefore, FLAP is an interesting pharmacological target to reduce LT biosynthesis during inflammatory processes. During the last years, FLAP gained importance as it was linked to cardiovascular diseases and a direct inhibition of 5-LOX was accompanied by adverse side effects.However, knowledge about the functionality and regulation of FLAP as well as its role in formation of pro-resolving lipid mediators is still limited. Furthermore, reliable data about FLAPs functionality under pathophysiological conditions are not available. Therefore, the main focus of this project is directed on FLAP in intact cells upon bacterial infection. First of all, physiologic stimulation conditions (pathogenic bacteria and their virulence factors) will be optimized in order to activate 5-LOX, induce lipid mediator (LM) biosynthesis while maintaining cell viability. UPLC-MS/MS will be applied to investigate LM biosynthesis with special focus on pro-resolving LM. Pharmacologic inhibition and knockdown of FLAP will be used to study the influence of FLAP on LM formation upon bacterial infection.In activated leukocytes, 5-LOX/FLAP complexes are assembled, which can be prevented by FLAP inhibitors. Part of this project will be the identification of additional potential 5-LOX-binding partners as dicer or coactosin-like protein (CLP) by using proximity ligation assay. Site-directed mutagenesis will be applied to identify and characterize the interaction site of 5-LOX and FLAP. HEK293 cells that co-express FLAP or its mutants with 5-LOX, will be investigated concerning complex assembly and LM biosynthesis. Since the activity of the related LTC4 synthase is decreased by phosphorylation, potential phosphorylation sites of FLAP will be investigated. The expected gain of knowledge about the functionality and regulation of FLAP in LM biosynthesis upon bacterial infection may provide new avenues to design inhibitors that interfere with the LT synthesis in order to effectively treat related inflammatory diseases.

• DFG Programme: Research Grants