Stabilisation and characterisation of the isolated regulatory domain of human 5-lipoxygenase |
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Authors: | Mirjam Schrö der,Ann-Kathrin Hä fner,Bettina Hofmann,Olof Rå dmark,Franz Tumulka,Rupert Abele,Volker Dö tsch,Dieter Steinhilber |
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Affiliation: | 1. Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany;2. Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institutet, S-17177 Stockholm, Sweden;3. Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany;4. Institute of Biophysical Chemistry, Goethe University Frankfurt, Max-von-Laue Strasse 9, D-60438 Frankfurt, Germany |
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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. |
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Keywords: | βME, β-mercaptoethanol BS3, bis[sulfosuccinimidyl]suberate BSA, bovine serum albumin CLP, coactosin-like protein cv, column volume DF, Dicer fragment containing amino acids 1650&ndash 1912 DPBS, Dulbecco's PBS DSF, differential scanning fluorimetry EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide Ig-CA, Igepal® CA-630 IPTG, isopropyl β-D-thiogalacto-pyranoside LOX, lipoxygenase LTA4, leukotriene A4 MBP, maltose-binding protein PC, phosphatidylcholine PLAT, polycystin-1, lipoxygenase, α-toxin SEC-MALS, size exclusion chromatography coupled with multi-angle light scattering detection s-NHS, N-hydroxysulfosuccinimide T20, Tween 20 TEV, tobacco etch virus protease Tm, melting temperature T0, reference melting temperature without additive ΔTm, change in melting temperature compared to reference melting temperature |
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