An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

Gerstmeier, Jana; Weinigel, Christina; Barz, Dagmar; Werz, Oliver; Garscha, Ulrike

doi:10.1016/j.bbagen.2014.05.016

Cited by 40 publications

(74 citation statements)

References 39 publications

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“…IF microscopy of 5-LO and FLAP in parallel enabled colocalization studies at the nuclear membrane (12,15,28), but whether the 2 proteins closely interact with each other cannot be answered using this technique. The PLA is a sophisticated method to reveal the proximity of 2 unmodified, native proteins (no tags, dyes, or labels) in situ of less than 40 nm distance, at a defined locale in the cell (24).…”

Section: Discussionmentioning

confidence: 99%

“…The DNA circle can be amplified and visualized by hybridization with a fluorophore (magenta). First, we utilized HEK293 cells that were stably transfected with recombinant FLAP and/or 5-LO and that were able to generate abundant 5-LO products when both 5-LO and FLAP are expressed (15). HEK293 cells were studied for colocalization of 5-LO and FLAP (Fig.…”

Section: In Situ Interaction Of 5-lo and Flapmentioning

confidence: 99%

“…HEK293 cells expressing FLAP and/or 5-LO were analyzed as described before (15). Neutrophils were preincubated with test compounds or vehicle for 10 min at 37°C resuspended in PBS, 0.1% glucose, 1 mM CaCl 2 and centrifuged onto poly-D-lysine coated glass coverslips (10 g for 2 s) prior stimulation.…”

Section: Analysis Of 5-lo Redistribution By Ifmentioning

confidence: 99%

“…HEK293 cells were cultured as monolayers and cells expressing FLAP and/or 5-LO were selected by 200 mg/ml hygromycin B and/or 400 mg/ml Geneticin, respectively, as described elsewhere (15).…”

Section: Cellsmentioning

confidence: 99%

“…It is assumed that FLAP acts as scaffold for 5-LO that governs the distribution of 5-LO to the perinuclear region, thus enabling efficient LTA 4 formation at this locale (14,15). The requirement of FLAP in intact cells is unique and indispensable for 5-LO product synthesis (but dispensable for other LOs), and FLAP appears to function as an arachidonic acid transfer protein for 5-LO, devoid of enzymatic activity (16,17).…”

mentioning

confidence: 99%

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Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

et al. 2015

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5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred by 5-LOactivating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca 2+ -ionophore A23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 mM) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.-Gerstmeier, J., Weinigel, C., Rummler, S., Rådmark, O., Werz, O., Garscha, U. Time-resolved in situ assembly of the leukotrienesynthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes. FASEB J. 30, 276-285 (2016). www.fasebj.org

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Section: Discussionmentioning

confidence: 99%

Section: In Situ Interaction Of 5-lo and Flapmentioning

confidence: 99%

Section: Analysis Of 5-lo Redistribution By Ifmentioning

confidence: 99%

Section: Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

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Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

et al. 2015

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Allosteric Activation of 15‐Lipoxygenase‐1 by Boswellic Acid Induces the Lipid Mediator Class Switch to Promote Resolution of Inflammation

et al. 2022

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Specialized pro‐resolving mediators (SPM), primarily produced in innate immune cells, exert crucial bioactions for resolving inflammation. Among various lipoxygenases (LOX), 15‐LOX‐1 is key for SPM biosynthesis, but cellular activation principles of 15‐LOX‐1 are unexplored. It was shown that 3‐O‐acetyl‐11‐keto‐β‐boswellic acid (AKBA) shifts 5‐LOX regiospecificity from 5‐ to 12‐lipoxygenation products. Here, it is demonstrated that AKBA additionally activates cellular 15‐LOX‐1 via an allosteric site accomplishing robust SPM formation in innate immune cells, particularly in M2 macrophages. Compared to ionophore, AKBA‐induced LOX activation is Ca2+‐ and phosphorylation‐independent, with modest induction of 5‐LOX products. AKBA docks into a groove between the catalytic and regulatory domains of 15‐LOX‐1 interacting with R98; replacement of R98 by alanine abolishes AKBA‐induced 15‐LOX product formation in HEK293 cells. In zymosan‐induced murine peritonitis, AKBA strikingly elevates SPM levels and promotes inflammation resolution. Together, targeted allosteric modulation of LOX activities governs SPM formation and offers new concepts for inflammation resolution pharmacotherapy.

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Identification of 2,4‐Dinitro‐Biphenyl‐Based Compounds as MAPEG Inhibitors

et al. 2022

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We identified 2,4‐dinitro‐biphenyl‐based compounds as new inhibitors of leukotriene C4 synthase (LTC4S) and 5‐lipoxygenase‐activating protein (FLAP), both members of the “Membrane Associated Proteins in Eicosanoid and Glutathione metabolism” (MAPEG) family involved in the biosynthesis of pro‐inflammatory eicosanoids. By molecular docking we evaluated the putative binding against the targets of interest, and by applying cell‐free and cell‐based assays we assessed the inhibition of LTC4S and FLAP by the small molecules at low micromolar concentrations. The present results integrate the previously observed inhibitory profile of the tested compounds against another MAPEG member, i. e., microsomal prostaglandin E2 synthase (mPGES)‐1, suggesting that the 2,4‐dinitro‐biphenyl scaffold is a suitable molecular platform for a multitargeting approach to modulate pro‐inflammatory mediators in inflammation and cancer treatment.

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An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

Cited by 40 publications

References 39 publications

Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

Allosteric Activation of 15‐Lipoxygenase‐1 by Boswellic Acid Induces the Lipid Mediator Class Switch to Promote Resolution of Inflammation

Identification of 2,4‐Dinitro‐Biphenyl‐Based Compounds as MAPEG Inhibitors

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