Time‐resolvedin situassembly 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-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

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

“…36) in A23187-challenged human neutrophils and monocytes as well as murine PMs from females, but not in male counterparts. Accordingly, pretreatment of female cells with 5α-DHT impeded 5-LO/FLAP interaction signals, effects that are typical for FLAP inhibitors (7). We conclude that the lower capacity of males to produce 5-LO products is due to androgens that can impede productive 5-LO/FLAP interactions and thus may hinder AA transfer via FLAP to 5-LO.…”

“…We thus hypothesized that a sex bias in 5-LO/ FLAP interaction, due to androgens, might underlie the different efficiencies of FLAP inhibitors and licofelone/Ssi. 5-LO/FLAP complex assembly at the nuclear membrane was studied using PLA that unravels in situ interactions of proteins that are in close proximity (<40 nm) (7,36). In resting human neutrophils and monocytes of both sexes, only sporadic 5-LO/FLAP signals were detectable ( Figure 5A).…”

“…To detect in situ interaction of 5-LO with FLAP in neutrophils, monocytes, or PMs, an in situ proximity ligation assay (PLA) (36) was performed as described before (7). Neutrophils were preincubated with 5α-DHT, MK-886, or vehicle for 10 minutes at room temperature (RT) in PGC buffer and centrifuged onto poly-d-lysine-coated glass coverslips (10 g for 2 seconds).…”

4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP)

“…In the first step of LT biosynthesis, 5-lipoxygenase (5-LO) catalyzes the production of the unstable epoxide LTA 4 from arachidonic acid (AA), which is further metabolized to LTB 4 or cysteinyl LTs (cys-LTs) such as LTC 4 , D 4 and E 4 [3,4]. This first step also requires the involvement of 5-LO-activating protein (FLAP), which acts as a regulatory protein by interaction with 5-LO for the transfer of AA to 5-LO for efficient metabolism [5].…”

“…Additionally, the 5-LO inhibitor setileuton (2, MK-0633) [7e9] and several FLAP inhibitors such as AM803 (3, GSK2190915), AZD6642 (4) and BI665915 (5) were reported to be in various stages of preclinical and clinical studies for treatment of asthma and COPD [10e13].…”