Graz University of Technology Austria Institute of Biochemistry ...

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.
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