title: Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes.
abstract: 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-LO-activating 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 µM) 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.
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jana
authors.initials: J
authors.lastname: Gerstmeier
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Christina
authors.initials: C
authors.lastname: Weinigel
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Silke
authors.initials: S
authors.lastname: Rummler
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden ulrike.garscha@uni-jena.de oliver.werz@uni-jena.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
authors.affiliation: *Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Jena, Germany; Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany; and Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, Stockholm, Sweden ulrike.garscha@uni-jena.de oliver.werz@uni-jena.de.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Garscha
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins / Q000378:metabolism*; D000328:Adult; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase
keywords: eicosanoids; inflammation; lipoxygenases; proximity-ligation assay