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for the determination of differences in enzyme patterns, e.g. due to effects of genetic background, environment, metabolic state and disease. Master Theses completed: Andreas Gasser: Determination of substrate- and stereoselectivity of lipases The elucidation of the reaction mechanisms of lipolytic enzymes requires the determination of the substrate- and stereoselectivities of these proteins. In order to find a more straightforward alternative to the common assays using radioactive substrates, a method using chiral, pyrenelabelled substrates was developed in this study, which allows the determination of substrateand stereoselectivity of lipases. This technique avoids the application of radioactive isotopes and as a consequence time consuming procedures. It is the basis for a simple and fast determination of lipase activity in high throughput analysis. On the basis of the robust and well characterized fungal Rhizomucor miehei lipase, the fluorescent substrates were tested for their applicability to determine substrate- and stereoselectivity of lipolytic enzymes using simple thin-layer chromatography. In order to obtain quantitative results and higher throughput, the method was adapted to HPLC analysis. This version was used to analyze activities as well as e.e. values of Rhizomucor miehei lipase, Chromobacterium viscosum lipase and Candida cylindracea cholesterol esterase. After overexpression in COS-7 cells, the selectivity and activity of the animal lipases adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and monoglyceride lipase (MGL) were measured in total cell lysates. Lipolytic activities were also determined in adipose tissue homogenates of ATGL- and HSLdeficient mice and compared to the wild type animals. The deficiency in the respective lipases correlated with the degradation patterns of the fluorescent substrates as determined by the above described method. Franziska Vogl: Transfection of macrophages with siRNA and plasmids. Expression and silencing of proteins targeted by oxidized phospholipids Oxidized phospholipids (oxPL) are generated from (poly)unsaturated glycerophospholipids in membranes and lipoproteins under conditions of oxidative stress. These compounds induce apoptosis in the cells of the vascular wall. In a previous functional proteomics study, the primary protein targets of a toxic aldehydophospholipid were identified in macrophages. To find out which of these protein candidates are involved in lipid-mediated cell death, we are following two different strategies. The first one uses siRNA technology to knock down the target proteins and measure the effect on cell death and signalling. The second approach is based on fusion proteins containing the protein candidates and RFP to measure Försterresonance-energy-transfer (FRET) and determine the spatial proximity to fluorescently labelled oxPL. In this diploma thesis, protocols for chemical transfection of RAW 264.7 cells with expression plasmids were optimized. Five fluorescent fusion proteins were cloned and expressed in Cos-7 cells. Chemical transfection and electroporation were used to transfect cells with siRNA against GAPDH and Caspase-3. Transfections and silencing were monitored using FACS-measurements, Western blotting and qPCR and proved to be efficient in both cases. Silencing of caspase-3 was associated with a decrease in protein expression and loss of function. As a consequence, enzyme knock-down rendered the cells more resistant towards oxPL-induced apoptosis, supporting the assumption that the protease is causally involved in programmed cell death. 30
International cooperations: T. Hianik, Department of Biophysics and Chemical Physics, Comenius University, Bratislava, Slovakia T. Hornemann, Institute for Clinical Chemistry, University Hospital Zürich; Zürich, Switzerland M. Hof, Department of Biophysical Chemistry, Academy of Sciences of the Czech Republic, Prague D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine; Cornell University, Ithaca, USA I. Parmryd, Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden T. Hugel, IMETUM, TU München, Garching, Germany P. Kinnunen, Biophysics, Aalto University, Helsinki, Finland Research projects: BM.W_F Genomforschung in Österreich (GEN-AU): Exploring the lipolytic proteome. Phospholipases (GOLD 3 project-Genomics of Lipid-associated Disorders, coordinated by KFU Graz). FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU Graz): Toxicity of oxidized phospholipids in macrophages. FWF Doctoral Program Molecular Enzymology: Functional enzyme analysis. FWF EUROCORES-EuroMEMBRANE-OXPHOS-Protein targets and apoptotic signaling of oxidized phospholipids Invited lectures: 1. Functional proteomic analysis of hydrolytic enzymes relevant to the stability and activity of oxidized phospholipids in macrophages. Cellular radical stress and related biomarkers, COST Chemistry CM0603, Athens, Greece, February 22, 2010 2. Molecular and supramolecular targets of oxidized phospholipids. ESF Euromembrane Meeting, Heidelberg, Germany, April 7, 2010 3. Primary targets of oxidized phospholipids in macrophages. 5 th Prague- Wroclaw Seminar on Biophysics, Prague, Czech Republic, November 18, 2010 Publications: 1. Landre JB, Blaess MF, Kohl M, Schlicksbier T, Ruryk A, Kinscherf R, Claus RA, Hermetter A, Keller M, Bauer M, Deigner HP Addressable Bipartite Molecular Hook (ABMH): Immobilized Hairpin Probes with Sensitivity below 50 Femtomolar Anal.Biochem. 397, 60-66, 2010. 2. Sattler W, Nusshold C, Kollroser M, Köfeler H, rechberger G, reicher H, Üllen A, Bernhart E,Waltl S,Kratzer I,Hermetter A,Hackl H, Trajanoski Z,Hrzenjak A, Malle E Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12neurons in vitro Free Radical Biol Med 48, 1588-1600, 2010. 31
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