title: Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis.
abstract: 5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden and.
authors.forename: Devaraj
authors.initials: D
authors.lastname: Basavarajappa
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden and.
authors.forename: Min
authors.initials: M
authors.lastname: Wan
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden and.
authors.forename: Ana
authors.initials: A
authors.lastname: Lukic
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, 60438 Frankfurt, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden and bengt.samuelsson@ki.se olof.radmark@ki.se.
authors.forename: Bengt
authors.initials: B
authors.lastname: Samuelsson
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden and bengt.samuelsson@ki.se olof.radmark@ki.se.
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D017997:Leukotriene C4 / Q000096:biosynthesis; D015289:Leukotrienes / Q000096:biosynthesis*; D008840:Microfilament Proteins / Q000378:metabolism*; D008954:Models, Biological; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; C118115:COTL1 protein, human; D007476:Ionophores; D015289:Leukotrienes; D008840:Microfilament Proteins; D016718:Arachidonic Acid; D017997:Leukotriene C4; D001094:Arachidonate 5-Lipoxygenase
keywords: eicosanoid; inflammation; oxylipin