Kühn Lab: Lipoxygenase Research
The cellular redox homeostasis is of major physiological relevance since the gene expression profile of most mammalian cells and thus their functional phenotype strongly depends on the redox state. Redox-sensitive transcription factors translate the redox equilibrium into gene expression alterations so that the cell can adapt to altered metabolic conditions. In mammalian cells redox homeostasis is maintained inter alia by the catalytic activities of pro- (lipoxygenases) and anti-oxidative (glutathione peroxidases) enzymes, which are the focus of our research.
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The cellular redoxHomöostasis is an important parameter determining the functional state of a cell. For a long time oxidative stress has been considered a deleterious process, which leads to oxidative modification of biomolecules and thus to functional impairment. However, during the past couple of years there has been a change in paradigm. Today it is generally accepted that normal cellular function requires a well-balanced redox homeostasis, which includes formation of reactive oxygen species. Expression of redox-sensitive genes is regulated by the cellular redox state.
In our group structure-activity relations of pro-oxidative Enzymes (Lipoxygenases) and anti-oxidative enzymes (Glutathionperoxidases) are investigated.
Lipoxygenases (LOX’s) are a heterogeneous group of lipid peroxydizing enzymes, which play an important role in cell differentiation, inflammatory and hyperproliferative diseases (1,2). Different isoforms are present in all higher developed animals and plants. LOX isoforms are classified according to their reaction specificity with polyunsaturated fatty acids as substrate. Within the human genome six different functional LOX genes are present, which encode for six different LOX isoforms. Although the crystal structure of different LOX isoforms were solved (1,2), the structural cause of different catalytic characteristics of LOX isoforms and their evolutionary relationships are widely unknown.
Main focus of our research is the investigation of the reaction mechanism of different LOX isoforms, phylogenetic aspects of LOX as lipid peroxidising enzymes and the physiological and patho-physiological relevance of LOX isoforms, which is investigated in different animal models of disease as well as in humans.
1. HaeggstroemJZ, FunkCD. Chem. Rev.111(2011)5866-5898.
2. IvanovI, HeydeckD, HofheinzK, RoffeisJ, O'DonnellVB, KuhnH, WaltherM. Arch. Biochem. Biophys. 503(2010)161-174.
Glutathioneperoxidases form a heterogenous family of peroxide reducing enzymes and utilize glutathione as electron donor (3). Intracellularly, they constitute the functional counterpart of lipoxygenases, which form fatty acid hydroperoxides. The human Genome contains several GPx-genes, some of which encode for selenocystein containing isoenzymes. The different GPx-isoforms differ from each other with respect to their cellular and subcellular lokalisation but also regarding their substrate specificity. Among the GPx-isoforms GPx4 is unique since it constitutes the only GPx-isoenzyme capable of reducing complex lipid hydroperoxides (phospholipid hydroperoxides, cholesterol ester hydroperoxides) even if these substrate are incoprporated within complex lipid-protein assemblies such as biomembranes, lipoproteins (4). Beside its role as peroxide reducing enzyme GPx4 also functions as structural protein because the mid-piece of sperms contains high amounts of GPx4 polymers.
3. Brigelius-FlohéR, MaiorinoM. Biochem Biophys Acta.1830(2013)3289-3303.
4. SchnurrK, BelknerJ, UrsiniF, ScheweT, KuehnH. J. Biol. Chem. 271(1996)4653-4658.
Our research strategy involves targeted protein modification (site directed mutagenesis) and characterization of the induced functional alterations. On the cellular level we explore the functional relevance of the above-mentioned proteins for the phenotype. This research includes experiments with gene-technically modified cells and organisms as well as inhibitor studies. Since an altered redox homeostasis has been implicated in the pathogenesis of various diseases (inflammation, atherosclerosis, adipositas) we will explore how overexpression and/or functional inactivation of the above mentioned proteins (lipoxygenases and glutathione peroxidases) impact disease development. To reach this goal we will employ transgenic mice that overexpress the ALOX15 gene in defined cells (macrophages, adipocytes). Alternatively, ALOX15 knockout and ALOX5 as well as GPx4 knockin mice will be employed, which lack expression of the corresponding gene or express the protein as inactive or altered enzyme mutant.
Crossing of these genetically manipulated mice should reveal the impact of cellular and systemic alterations in the peroxide turnover. The physiological and patho-physiological consequences of these gene-technical modifications will be investigated in several animal disease models (e.g. DSS-induced colitis) to explore the impact of peroxide turnover in the pathogenesis of selected disorders.