Stabilisation and characterisation of the isolated regulatory domain of human 5-lipoxygenase

• Published in: Biochimica et biophysica acta 2014-10, Vol.1841 (10), p.1538-1547
• Main Authors: Schröder, Mirjam, Häfner, Ann-Kathrin, Hofmann, Bettina, Rådmark, Olof, Tumulka, Franz, Abele, Rupert, Dötsch, Volker, Steinhilber, Dieter
• Format: Article
• Language: English
• Subjects: Aggregation; Coactosin-like protein; Dicer; Differential scanning fluorimetry; Lipoxygenase; Medicin och hälsovetenskap; PLAT
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2014.07.022

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. [Display omitted] •The isolated regulatory PLAT domain of 5-lipoxygenase forms soluble aggregates.•Mutation W75G reduces aggregation and thermal instability of the isolated domain.•Stabilisation of the isolated regulatory domain enables mechanistic studies.•The isolated PLAT W75G interacts with Dicer, but not with coactosin-like protein.•DSF of the isolated domain allows future discovery of novel allosteric inhibitors.