Journal
FASEB JOURNAL
Volume 30, Issue 5, Pages 1892-1900Publisher
FEDERATION AMER SOC EXP BIOL
DOI: 10.1096/fj.201500210R
Keywords
arachidonic acid; HEK293; inflammation; leukotriene A(4); translocation
Categories
Funding
- Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Centres: Chemical Mediators in complex Biosystems [SFB 1127]
- U.S. National Institutes of Health, National Heart, Lung, and Blood Institute [HL 107887]
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Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA(4). 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (approximate to 60-70% 5-LOX-wt levels) but not of LTA(4) hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p) ETE at levels comparable to 5-LOX-wt but reduced LTA(4) hydrolysis products. Coexpression of FLAP partially restored LTA(4) hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. www.fasebj.org
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