Studies on coactosin-like protein interaction with 5-lipoxygenase

Sammanfattning: Leukotrienes (LTs) are proinflammatory lipid mediators derived from polyunsaturated arachidonic acid, which is ester-bound at the sn-2 position of glycerol in cellular phospholipid bilayers. In LTs biosynthesis, 5-lipoxygenase (5-LO) represents the key enzyme, which catalyzes two initial steps. The aim of present thesis was to investigate the effects of a small 5-LO binding protein named Coactosin-like Protein (CLP) on the 5-LO activation and stability in order to define the potential role of CLP in mechanisms involved in formation of LTs. First, we found that NMR structure of human CLP is composed of a five-stranded b-sheet surrounded by four alpha-helices. The structure also revealed high flexibility of the C-terminus of CLP molecule and the loop connecting beta3 and beta4. Surface-exposed Lys-75, a critical binding residue for F-actin, and Lys-131, essential residue for CLP binding to 5-LO are shown to be close to each other, precluding simultaneous binding of F-actin and 5-LO to CLP. The effects of CLP on the 5-LO activation and modulation of its enzymatic product profile were assessed in 5-LO activity assays in vitro. We have found three major CLP-stimulated effects on the 5-LO catalytic reaction. First, CLP upregulates Ca2+/Mg+-induced 5-LO activity in the absence of phosphatidylcholine (membrane). In such incubations, formation of 5-HETE is found to be highly increased, while the production of leukotriene A4 (LTA4) is minute. These data suggest that CLP can function as a scaffold for 5-LO similar to membranes. Second, CLP increases the amount of LTA4 formed, by approximately 3-fold, when present together with phosphatidylcholine. Third, CLP promotes formation of 5-hydroxyeicosatetraenoic acid (5-HETE) by substantial reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which results in an increased ratio of 5-HETE/5-HPETE independently of the presence of a membrane. CLP also stabilizes 5-LO and prevents its non-turnover inactivation, which together with the effects on the 5-LO activity, suggests that CLP might be functioning as a chaperone of 5-LO. Trp-102, positioned in the beta-sandwich of the 5-LO structure, is required for binding of CLP to 5-LO and for all the above-mentioned CLP-induced effects on 5-LO activity and its structural stabilization. Therefore, the beta-sandwich appears to be the essential part of the CLP binding surface on the 5-LO molecule. The same pattern of subcellular trafficking (nuclear/nonnuclear association) was found for 5-LO and CLP upon activation of human polymorphonuclear leukocytes and Mono Mac 6 cells induced by different cell stimuli. This finding indicates an association between 5-LO and CLP in the cell, and combined with the results from the 5-LO activity assays, it also implies an important role of CLP in upregulation of LT biosynthesis at the nuclear membrane. Hyperforin, an anti-inflammatory compound isolated from St. John's wort and previously reported to inhibit 5-LO activity both in vitro and in cellular systems, successfully interrupted formation of functional 5-LO:CLP complex. Hyperforin reduced the binding of 5-LO to CLP at concentrations shown to suppress synthesis of the 5-LO products, implying its mode of action by interfering with the 5-LO beta-sandwich. Taken together, the findings presented in this thesis have pinpointed CLP as a novel player in the 5-LO signaling pathway, capable of activating 5-LO enzyme and modulating its product pattern. Thus, these results have also potentially opened an interesting window for therapeutic intervention on LT biosynthesis.