The nuclear membrane leukotriene synthetic complex is a signal integrator and transducer

Bair, Angela M.; Turman, Melissa V.; Vaine, Christine A.; Panettieri, Reynold A.; Soberman, Roy J.

doi:10.1091/mbc.e12-06-0489

Cited by 35 publications

(39 citation statements)

References 41 publications

(48 reference statements)

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“…Furthermore, earlier studies discussed the regulatory role of endogenously released arachidonic acid for 5-LO translocation (47). We show that cPLA 2 inhibition prevents 5-LO/FLAP interaction, which is in agreement with previous studies (28), and supplementation of arachidonic acid or 5-HPETE restored the 5-LO/FLAP complex. Hence, arachidonic acid or 5-HPETE is essential for the 5-LO/FLAP interaction, possibly by acting as adaptor molecules.…”

Section: Discussionsupporting

confidence: 92%

“…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%

“…5-LO activity, 5-LO translocation, and interaction of 5-LO with FLAP in these leukocytes are commonly analyzed 10 to 30 min upon stimulation (20,23,28), but the exact time course and correlation of these processes and are unknown. Therefore, we assessed the subcellular localization of 5-LO and FLAP in monocytes ( Fig.…”

Section: Time-resolved Colocalization and Interaction Of 5-lo And Flamentioning

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: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Time-resolved Colocalization and Interaction Of 5-lo And Flamentioning

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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“…2C), indicating that during this mimic of physiological conditions, all detectable 5LO activity occurred at the nuclear membrane. Furthermore, our finding that FLAP knockdown reduced the yield of LTC 4 in LTA 4 incubations indicates that FLAP may support the activity of LTC 4 synthase, in line with these two proteins in the membrane-associated proteins in eicosanoid and glutathione metabolism family forming a cellular complex (5,22).…”

Section: Discussionmentioning

confidence: 56%

Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis

Basavarajappa

Wan

Lukić

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…It has recently been shown that leukotriene synthesis involves activationdependent formation of supramolecular complexes on the nuclear envelope composed of FLAP with 5-LO and FLAP with leukotriene C 4 synthase ( 117,118 ). In addition, cPLA 2 ␣ -derived arachidonic acid was shown to affect the conformation of FLAP and enhance its ability to recruit 5-LO for complex formation ( 119 ). It is not known whether cPLA 2 ␣ physically associates with these complexes or whether arachidonic acid released in their vicinity diffuses through the membrane to initiate leukotriene production.…”

Section: Downloaded Frommentioning

confidence: 99%

Cytosolic phospholipase A2: physiological function and role in disease

Leslie

2015

Journal of Lipid Research

330

257

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The nuclear membrane leukotriene synthetic complex is a signal integrator and transducer

Cited by 35 publications

References 41 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

Roles of coactosin-like protein (CLP) and 5-lipoxygenase-activating protein (FLAP) in cellular leukotriene biosynthesis

Cytosolic phospholipase A2: physiological function and role in disease

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