Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP.
Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Ann-Kathrin
AK
Häfner
Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena, Germany.
Jana
J
Gerstmeier
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Michael
M
Hörnig
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Sven
S
George
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Ann-Katrin
AK
Ball
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
Mirjam
M
Schröder
Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena, Germany.
Ulrike
U
Garscha
Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena, Germany.
Oliver
O
Werz
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Electronic address: steinhilber@em.uni-frankfurt.de.
Dieter
D
Steinhilber
D058927:5-Lipoxygenase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001665:Binding Sites; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003958:Diamide / Q000494:pharmacology; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015289:Leukotrienes / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D061251:Primary Cell Culture; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D015289:Leukotrienes; D011994:Recombinant Proteins; D003958:Diamide; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase; C583212:ALOX5 protein, human; D005978:Glutathione
FLAP; Inflammation; Leukotrienes; Lipoxygenase; Subcellular Localization