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

白三烯（lekotrienes， LT）是一种促炎介质，不仅与慢性炎症性疾病有关，还与阿尔茨海默病、癌症和心血管疾病有关。因此，LT生物合成的药物干预是一种很有前景的治疗策略。LT生物合成的第一步是由花生四烯酸（AA）通过5-脂氧合酶（5-LOX）形成LTA4。受到刺激后，5-LOX通过多种机制被激活，并易位到核膜上，其中5-LOX激活蛋白（FLAP）位于核膜上。敲除研究和药理学抑制FLAP完全消除完整白细胞中的LT生物合成。因此，FLAP是炎症过程中减少LT生物合成的一个有趣的药理学靶点。在过去的几年里，FLAP变得越来越重要，因为它与心血管疾病有关，并且直接抑制5-LOX伴随着不良副作用。然而，关于FLAP的功能和调控及其在促溶解脂质介质形成中的作用的知识仍然有限。此外，关于FLAPs在病理生理条件下功能的可靠数据是不可用的。因此，本项目的主要重点是针对完整细胞在细菌感染时的FLAP。首先，优化生理刺激条件（病原菌及其毒力因子），激活5-LOX，诱导脂质介质（LM）生物合成，同时维持细胞活力。UPLC-MS/MS将应用于LM生物合成研究，重点研究前分解LM。我们将利用药物抑制和敲低FLAP来研究FLAP对细菌感染后LM形成的影响。在活化的白细胞中，5-LOX/FLAP复合物被组装，这可以被FLAP抑制剂阻止。该项目的一部分工作将是通过近距离结联试验确定其他潜在的5- lox结合伙伴，如dicer或coactosin样蛋白（CLP）。位点定向诱变将用于鉴定和表征5-LOX和FLAP的相互作用位点。将研究与5-LOX共表达FLAP或其突变体的HEK293细胞在复合物组装和LM生物合成方面的作用。由于相关LTC4合成酶的活性会因磷酸化而降低，因此我们将研究FLAP的潜在磷酸化位点。对细菌感染时LM生物合成中FLAP的功能和调控的预期获得可能为设计干扰LT合成的抑制剂提供新的途径，从而有效治疗相关炎症疾病。

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.