Studies on coactosin-like protein interaction with 5-lipoxygenase

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><h3>List of scientific papers</h3><p>I. Liepinsh E, Rakonjac M, Boissonneault V, Provost P, Samuelsson B, Rådmark O, Otting G (2004). NMR structure of human coactosin-like protein. J Biomol NMR. 30(3): 353-6. <br><a href="https://doi.org/10.1007/s10858-004-3449-y">https://doi.org/10.1007/s10858-004-3449-y</a><br><br> </p><p>II. Rakonjac M, Fischer L, Provost P, Werz O, Steinhilber D, Samuelsson B, Rådmark O (2006). Coactosin-like protein supports 5-lipoxygenase enzyme activity and up-regulates leukotriene A4 production. Proc Natl Acad Sci USA. 103(35): 13150-5. <br><a href="https://doi.org/10.1073/pnas.0605150103">https://doi.org/10.1073/pnas.0605150103</a><br><br> </p><p>III. Esser J, Rakonjac M, Hofmann B, Fischer L, Provost P, Schneider G, Steinhilber D, Samuelsson B, Rådmark O (2009). Coactosin-like protein functions as a stabilizing chaperone for 5-lipoxygenase: role of tryptophan 102. Biochem J. [Accepted] <br><a href="https://doi.org/10.1042/BJ20090856">https://doi.org/10.1042/BJ20090856</a><br><br> </p><p>IV. Feisst C, Pergola C, Rakonjac M, Rossi A, Koeberle A, Dodt G, Hoffmann M, Hoernig C, Fischer L, Steinhilber D, Franke L, Schneider G, Rådmark O, Sautebin L, Werz O (2009). Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo. Cell Mol Life Sci. 66(16): 2759-71. <br><a href="https://doi.org/10.1007/s00018-009-0078-3">https://doi.org/10.1007/s00018-009-0078-3</a><br><br> </p>