Abstract
5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.
Highlights
Lipoxygenases (LOXs) catalyze the regio- and stereospecific insertion of molecular oxygen into polyunsaturated fatty acids [1]
To assess whether these effects can be extended to the human LOXs, and to gain insight into the mechanism(s) by which iron may modulate 5-LOX membrane binding, we investigated by fluorescence resonance energy transfer (FRET) the membrane binding properties of apo5-LOX and holo-5-LOX
We found that iron removal induced a significant decrease (∼2fold over the holo-5-LOX) in the affinity of the apo-form of the enzyme for POPC membranes, as indicated by an increase in [L]1/2 (49.4 ± 2.8 μM) of the apo-enzyme with respect to the value calculated for holo-5-LOX (Figure 1B)
Summary
Lipoxygenases (LOXs) catalyze the regio- and stereospecific insertion of molecular oxygen into polyunsaturated fatty acids [1]. In peripheral blood neutrophils [7], differentiated HL-60 cells [8], and peritoneal macrophages [9], 5LOX is mainly localized in the cytosol, whereas in resting alveolar macrophages [10], rat basophilic leukemia cells [11], bone marrow–derived mast cells [12], and Langerhans cells of human skin [13], the same enzyme is either partly or predominantly present in the soluble compartment of the nucleus Upon stimulation, both cytosolic and nuclear 5-LOXs translocate to the nuclear envelope, leading to interaction with the 5-LOX activating protein (FLAP), a small protein localized in internal cell membranes that is essential in the functional processing of endogenous AA [3, 14]. The translocation from the cytosol to the nuclear membrane of 5-LOX and its colocalization with FLAP is clearly emerging as an early and ratelimiting mechanism of activation that triggers different signaling pathways leading to the synthesis of different classes of proinflammatory LTs (LTA4 and LTC4) [14, 15]
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