title: Stabilisation and characterisation of the isolated regulatory domain of human 5-lipoxygenase.
abstract: 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.
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Schröder
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Häfner
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Hofmann
authors.affiliation: Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-17177 Stockholm, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Tumulka
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Rupert
authors.initials: R
authors.lastname: Abele
authors.affiliation: Institute of Biophysical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany. Electronic address: steinhilber@em.uni-frankfurt.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
keywords: Aggregation; Coactosin-like protein; Dicer; Differential scanning fluorimetry; Lipoxygenase; PLAT