GEN-AU GOLD II (Genomics of Lipid-associated Disorders): C4 - Functional and differential analysis of lipolytic enzymes by affinity tagging

  • Schicher, Maximilian, (Teilnehmer (Co-Investigator))
  • Zenzmaier, Elfriede (Teilnehmer (Co-Investigator))
  • Bobula, Tomas, (Teilnehmer (Co-Investigator))
  • Lehsl, Annemarie, (Teilnehmer (Co-Investigator))
  • Morak, Maria, (Teilnehmer (Co-Investigator))
  • Dunai, Zsuzsanna, (Teilnehmer (Co-Investigator))
  • Birner-Grünberger, Ruth (Projektleiter (Principal Investigator))
  • Hermetter, Albin (Projektleiter (Principal Investigator))

Projekt: Foschungsprojekt

Beschreibung

In the postgenomic era, researchers are now challenging the proteome with methods like two- dimensional gel electrophoresis or multidimensional chromatography followed by mass spectrometry and various other methods for abundance-based proteome profiling. However, the amount of proteins present at a certain state of a cell does not always correlate with enzyme activities responsible for the metabolic fluxes, cell management and signal transduction. Therefore, elucidation of changes in protein activity is the ultimate goal of proteomics.
Our laboratory has been developing and using fluorescently labeled phosphonic acid esters as suicide inhibitors for selective detection of lipolytic enzymes (lipases, esterases) in complex proteomes. These biocatalysts hydrolyze carboxylic acid esters, thioesters and amides inside and outside cells thus fulfilling specific functions in lipid metabolism, degradation, mobilization and signaling.
The enzyme-specific phosphonate inhibitors covalently react with the nucleophilic serine in the active site of lipases and esterases. This reaction is stoichiometric and irreversible. Stable lipid-protein complexes are formed that can be detected on the basis of their fluorescence signal after electrophoretic separation from inactive proteins on polyacrylamide gels eventually followed by mass spectroscopic identification. The enzyme-specific probes were successfully used for functional analysis of crude lipase preparations, the lipolytic proteome of mouse adipose tissue and other tissues. In the future, a combination of specifically designed enzyme probes will be developed for comparative activity profiling of closely related proteomes to determine changes in enzyme activities due to differences in genetic background, environment and subcellular localization with high precision.
StatusAbschlussdatum
Tatsächlicher Beginn/ -es Ende1/12/0530/11/08