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5-Lipoxygenase (5-LOX) is the key player of pro-inflammatory leukotriene biosynthesis. Its regulatory or so-called PLAT (polycystin-1, lipoxygenase, α-toxin) domain binds allosteric modulators like calcium, membranes, coactosin-like protein and Dicer, thereby influencing 5-LOX activity at the nuclear membrane by mediating translocation. The PLAT domain may also regulate cytosolic 5-LOX activity and possibly influence microRNA metabolism. Hence, it has also evolved as a promising target for anti-inflammatory therapy. Research focusing on this substructure of 5-LOX requires an assay system based on the isolated domain. However, we found that the isolated PLAT domain was highly prone to aggregation and therefore unsuitable for interaction studies. Substitution of the single, membrane-binding tryptophan 75 with glycine reduced aggregation and substantially increased its thermal stability. Calcium interaction of the single mutant was confirmed by differential scanning fluorimetry. Moreover, crosslinking experiments demonstrated the ability of the isolated PLAT domain to bind Dicer C-terminus whereas the interaction with coactosin-like protein required the interplay of the catalytic and the PLAT domain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]