Staff Members of the Institute of Biochemistry, TU - Institut für ...

Doctoral Theses completed:
Maria Morak: Functional proteomic analysis and comparison of lipolytic enzymes in mouse
tissues
Lipases and esterases catalyze the degradation of carboxylic acid esters including mono-, di-,
and triacylglycerols, and cholesteryl esters. The hydrolysis of these substrates is a key process
in energy homeostasis, lipid remodeling and signaling of eucaryotes. Disorders of lipid
homeostasis may lead to obesity, type 2 diabetes, atherosclerosis, dyslipidaemia and other
lipid-associated diseases. In order to identify the active lipolytic enzymes in complex
proteomes of mouse tissues, fluorescent and biotinylated p-nitrophenyl phosphonic acid esters
were used as activity-recognition probes for selective protein tagging followed by
electrophoretic separation and analysis by nanoHPLC-MS/MS. In addition, differential
activity-based gel electrophoresis (DABGE) was developed as a novel technique for the
comparison of lipolytic enzymes from two different biological samples in one gel. This
method was applied to compare the lipolytic proteomes of brown and white adipose tissue
from wild-type mice and animals deficient in the key enzymes responsible for intracellular
TAG hydrolysis in these organs. We found characteristic lipase patterns in BAT and WAT
that specifically responded to the expression of ATGL (adipose triglyceride lipase) and HSL
(hormone-sensitive lipase).
Maximilian Schicher: Functional proteomic analysis of lipolytic enzymes in cultured murine
and human fat cells
Lipolytic enzymes are responsible for intra- and extracellular degradation of acylglycerols and
cholesterol esters. The hydrolysis of these compounds is a key event in energy homeostasis
and signaling processes in mammals and humans. Impairment of lipid metabolism may lead
to diseases such as insulin resistance, obesity, type 2 diabetes, atherosclerosis and other lipidassociated
disorders. We used a functional proteomics approach based on fluorescent and
biotinylated p-nitrophenyl phosphonates as activity-based probes to identify active lipolytic
and esterolytic enzymes in subcutaneous and visceral human adipocytes as well as
undifferentiated and differentiated murine fat cells. The labeled proteins were separated by gel
electrophoresis, imaged with a laser scanner and identified by LC-MS/MS after in-gel
digestion. Furthermore, we compared the lipolytic activities of subcutaneous and visceral
adipocytes in a single polyacrylamide gel by differential activity-based gel electrophoresis.
We found specific enzyme patterns depending on the differentiation state and localization of
fat cells. The functional analysis of overexpressed proteins revealed novel lipase candidates.
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
M. Palmer, Department of Chemistry, University of Waterloo, Ontario, Canada
D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine;
Cornell University, Ithaca, USA
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