title: 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.
abstract: 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 LTA4 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 (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 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.
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and.
authors: Jana
authors: J
authors: Gerstmeier
authors: Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA.
authors: Marcia E
authors: ME
authors: Newcomer
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and.
authors: Sophie
authors: S
authors: Dennhardt
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and.
authors: Erik
authors: E
authors: Romp
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and.
authors: Jana
authors: J
authors: Fischer
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and.
authors: Oliver
authors: O
authors: Werz
authors: Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany; and ulrike.garscha@uni-jena.de.
authors: Ulrike
authors: U
authors: Garscha
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000502:physiology*; D002465:Cell Movement; D002467:Cell Nucleus; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D002722:Chlorobenzenes
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase; D007211:Indoles; C060893:MK-886; D002722:Chlorobenzenes
keywords: HEK293; arachidonic acid; inflammation; leukotriene A4; translocation