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 A 4 to leukotriene C 4 (LTC 4 ), suggesting a role for the activity of LTC 4 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.inflammation | eicosanoid | oxylipin L eukotrienes (LTs) are lipid mediators of inflammation with roles in both normal host defense and pathophysiological conditions (1). 5-Lipoxygenase (5LO) is a key enzyme in LT biosynthesis, and its regulation is a determinant of LT formation (2). In addition, 5LO is involved in biosynthesis of lipoxins and resolvin E1 (3). Cellular 5LO activity depends on several cofactors that regulate LT biosynthesis and prevent LT production in unstimulated situations. Increased intracellular Ca 2+ levels activate and translocate 5LO to the perinuclear region, where it accesses its substrate arachidonic acid (AA) released by cytosolic phospholipase A 2 (cPLA 2 ) (1). Cellular formation of LTs from endogenous AA depends on the small membrane protein 5LO-activating protein (FLAP) (4). FLAP has been suggested to function as a membrane anchor scaffold for 5LO to facilitate the transfer of AA to 5LO (5,6). In the cell, 5LO is subject to phosphorylation. MAP kinase pathways can activate 5LO during cell stress (7), whereas phosphorylation by protein kinase A inhibits cellular 5LO activity (8).5LO has two domains, an N-terminal C 2 -like regulatory domain with a β-sandwich structure and a catalytic C-terminal domain composed predominantly of α-helices that harbors the prosthetic iron (9). C...

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males

“…Pharmacological and genetic approaches in vitro and in vivo revealed the strong requirement of FLAP for cellular LT biosynthesis (4,27,47,48). FLAP inhibitors reduce LT biosynthesis by preventing the interaction between FLAP and 5-LO at the nuclear membrane (7), thereby blocking AA transfer from FLAP to 5-LO (5).…”

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

“…Mouse anti-5-LO monoclonal antibody was a generous gift by Dr. Dieter Steinhilber (Goethe University, Frankfurt, Germany). Rabbit anti-CLP polyclonal antibody was from Acris Antibodies (San Diego, CA, USA); rabbit anti-FLAP polyclonal antibody, Abcam (Cambridge, United Kingdom); mouse anti-actin monoclonal antibody, Santa Cruz (Heidelberg, Germany); tritium-labeled [5,6,8,9,11,12,14, H]AA, Biotrend Chemicals GmbH (Cologne, Germany); HPLC solvents, Merck (Darmstadt, Germany); poly-D-lysin coated glass coverslips, neuVitro (El Monte, CA, USA). Duolink detection reagents red, Duolink PLA (proximity ligation assay) probe anti-rabbit PLUS, Duolink PLA probe antimouse MINUS, as well as Duolink wash buffers and all other chemicals were from Sigma-Aldrich (Deisenhofen, Germany) unless stated otherwise.…”

“…First, 5-LO catalyzes the incorporation of molecular oxygen at C5 of arachidonic acid yielding the intermediate 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) that in the second step is dehydrated by 5-LO to LTA 4 (5). The cellular capacity to generate 5-HPETE and LTA 4 depends on the translocation of 5-LO from a soluble compartment to the nuclear membrane, regulated by a fine-tuned machinery involving Ca 2+ -mediated binding to phosphatidylcholine (PC) in the nuclear membrane, phosphorylation at serine residues (6)(7)(8)(9)(10), and interaction of 5-LO with coactosin-like protein (CLP) (11,12).…”

“…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). Genetic knockdown or inhibition of FLAP prevents LT formation in cell-based assays and in vivo, and FLAP inhibitors were reported to interfere with nuclear translocation of 5-LO (12,18 3 H-labeled arachidonic acid; 5-LO, 5-lipoxygenase; 5-HPETE, 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid; cPLA 2 a, cytosolic phospholipase A 2 a; CLP, coactosin-like protein; FLAP, 5-LO-activating protein; IF, immunofluorescence microscopy; LT, leukotriene; PC, phosphatidylcholine; PLA, proximity ligation assay proposed interaction between 5-LO and FLAP in intact human leukocytes, concomitant to 5-LO product synthesis, is still obscure and if additional factors regulate this interaction is elusive. Despite in vitro pull-down assays with artificial GST-tagged FLAP and 5-LO (19), coimmunoprecipitation experiments (20), or association studies using membrane-permanent cross-linking agents to perform fluorescence lifetime imaging microscopy in mouse neutrophils and rat RBL-2H3 cell lines (20), there has been no direct proof for an interaction of the 2 native proteins in intact human leukocytes, until now.…”

Tandem Benzophenone Amino Pyridines, Potent and Selective Inhibitors of Human Leukotriene C4 Synthase

“…LTs, 5-HETE, and lipoxins were obtained from the aliquots (2 ml) of the stopped reaction mixtures by SPE before reversed-phase HPLC as described before (Basavarajappa et al, 2014). Prostaglandin B 2 (100 pmol) and 17-OH-C22:4 (100 pmol) were added as internal standards, kind gifts from Mats Hamberg (Karolinska Institutet).…”