Distinct parts of leukotriene C(4) synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein.
Leukotriene C(4) is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C(4) synthase (LTC(4)S) participate in its biosynthesis. We report evidence that LTC(4)S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC(4)S. FLAP bound to the N-terminal part/first hydrophobic region of LTC(4)S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC(4)S. Fluorescent FLAP- and LTC(4)S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC(4)S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC(4)S. Direct interaction of 5-LO and LTC(4)S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC(4)S.
Strid
Tobias
T
Department of Clinical and Experimental Medicine, Linköping University, Sweden.
Svartz
Jesper
J
Franck
Niclas
N
Hallin
Elisabeth
E
Ingelsson
Björn
B
Söderström
Mats
M
Hammarström
Sven
S
D058927:5-Lipoxygenase-Activating Proteins; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase