Coactosin-Like Protein Supports 5-Lipoxygenase Enzyme Activity and up-Regulates Leukotriene A₄ Production

• Published in: Proceedings of the National Academy of Sciences - PNAS 2006-08, Vol.103 (35), p.13150-13155
• Main Authors: Rakonjac, Marija, Fischer, Lutz, Provost, Patrick, Werz, Oliver, Steinhilber, Dieter, Samuelsson, Bengt, Rådmark, Olof
• Format: Article
• Language: English
• Subjects: Arachidonate 5-Lipoxygenase - metabolism; Biochemistry; Biological Sciences; Biosynthesis; Calcium - pharmacology; Catalysis - drug effects; Cell Nucleus - drug effects; Enzyme activity; Humans; Ionophores - pharmacology; Kinetics; Leukocytes; Leukocytes - drug effects; Leukocytes - enzymology; Leukotriene A4 - biosynthesis; Leukotrienes; Lipid Peroxides - metabolism; Magnesium - pharmacology; Microfilament Proteins - metabolism; Mutant Proteins - metabolism; Nuclear membrane; Peroxidases - metabolism; Protein Binding - drug effects; Protein Transport - drug effects; Proteins; Scaffolds; Stoichiometry; Time Factors; Total product; Tryptophan - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0605150103

Regulation of 5-lipoxygenase (5LO) activity is a key determinant for the biosynthesis of proinflammatory leukotrienes. Coactosinlike protein (CLP) is an F-actin-binding protein that can also bind 5LO. Here, we report that CLP can up-regulate and modulate 5LO activity [formation of 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE)], 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HETE), and 5(S)-trans-5,6-oxido-7,9-trans-11,14cis-eicosatetraenoic acid (LTA₄) in vitro. Three findings are presented. First, CLP up-regulates Ca²⁺ -induced 5LO activity, in the absence of phosphatidylcholine (membrane). Apparently, CLP can function as a scaffold for 5LO, similar to membranes. Second, CLP gives a considerable (3-fold) increase in the amount of LTA₄ formed by 5LO, when present together with phosphatidylcholine. Third, CLP increases the ratio of 5-HETE/5-HPETE. These effects require protein interaction by Trp residues in ligand-binding loops of the 5LO β-sandwich; both binding and stimulatory effects of CLP were abolished for the mutant 5LO-W13/75/102A. In polymorphonuclear leukocytes stimulated with Ca²⁺ ionophore, both CLP and 5LO associated with the nucleus, whereas in resting cells, CLP and 5LO were cytosolic. These findings establish CLP as a factor relevant for 5LO product formation. Functioning as a 5LO scaffold, CLP may provide a basis for the formation of 5-HETE in the cytosol of different cell types. Furthermore, in stimulated cells, CLP appears to function in a complex together with 5LO and membranes, increasing the capacity of 5LO for leukotriene biosynthesis.