Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes
| Autor: | Ulrike Garscha, Oliver Werz, Silke Rummler, Christina Weinigel, Jana Gerstmeier, Olof Rådmark |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Adult
0301 basic medicine 5-Lipoxygenase-Activating Proteins Proximity ligation assay Biochemistry Cell membrane 03 medical and health sciences chemistry.chemical_compound Leukocytes Genetics medicine Humans 5-lipoxygenase-activating protein Molecular Biology Cells Cultured Leukotriene Arachidonate 5-Lipoxygenase Arachidonic Acid biology Cell Membrane Cell biology Transport protein Protein Transport HEK293 Cells 030104 developmental biology medicine.anatomical_structure chemistry Arachidonate 5-lipoxygenase biology.protein Female Arachidonic acid Cell activation Protein Binding Biotechnology |
| Zdroj: | The FASEB Journal. 30:276-285 |
| ISSN: | 1530-6860 0892-6638 |
| DOI: | 10.1096/fj.15-278010 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text