1. |
- Rakonjac, Marija
(author)
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Studies on coactosin-like protein interaction with 5-lipoxygenase
- 2009
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Doctoral thesis (other academic/artistic)abstract
- 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.
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2. |
- Ryge, Marija Rakonjac, et al.
(author)
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A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity
- 2014
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 545, s. 179-185
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Journal article (peer-reviewed)abstract
- 5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like beta-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the beta-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (v(init)) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix alpha 2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like beta-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX. (C) 2014 Elsevier Inc. All rights reserved.
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3. |
- Woolcott, Orison O, et al.
(author)
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Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells.
- 2006
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In: Cell Calcium. - : Elsevier BV. - 0143-4160 .- 1532-1991. ; 39:6, s. 529-37
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Journal article (peer-reviewed)abstract
- Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.
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4. |
- Zhou, Chunfang, et al.
(author)
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Morphological properties of nanoporous folic acid materials and in vitro assessment of their biocompatibility
- 2012
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In: Nanomedicine. - : Future Medicine Ltd. - 1743-5889 .- 1748-6963. ; 7:3, s. 327-334
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Journal article (peer-reviewed)abstract
- Background: Mesoporous silica-based particles are of potential interest for the development of novel therapeutic targeted delivery vehicles. Their ability to load and release large quantities of active pharmaceutical products with varying properties, combining controlled and targeted release functions make them unique amongst nanotechnology-based carrier systems.Materials & methods: In this study, nanoporous folic acid-templated materials (NFM-1) were prepared and the synthetic strategies for the control of textural and morphology properties of NFM-1 are described. The potential biocompatibility of NFM-1 particles with different morphology (gyroid shaped, fibers and rod-shaped) was assessed using a panel of human cell lines.Results: The results reveal that NFM-1 morphology has an impact on cell viability such that particles showing higher aspect ratios possess increased cytotoxicity.Conclusion: These studies provide useful information for the development of novel mesoporous materials for biomedical applications, including cell-specific drug delivery.
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