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A two-dimensional map and database of soluble nuclear proteins from HepG2 cells as reference for identification of
H Baumeister1, M Schärfke, S Haebel
1Nemod GmbH, Berlin, Germany. hans.baumeister@nemod.com
Background:
Eukaryotic cells of higher organisms are able to regulate gene transcription in response to changes in the supply of nutrients. In hepatocytes, extracellular glucose levels affect transcription of genes that encode enzymes engaged in glycolysis, gluconeogenesis and lipogenesis. While glucose response elements have been located within a few model gene promoters, the identity of glucose-sensing transcription factors and the mechanisms of their activation remain to be elucidated.
Aim Of The Study:
We intended to establish a two-dimensional map of nuclear proteins as a reference for identification of nutrient-regulated transcription factors.
Methods:
Human hepatoma HepG2 cells were used for the preparation of nuclear extracts. 150-200 microg of the protein mixture were analyzed by 2-dimensional gel electrophoresis (2-DE) and silver-stained protein spots were identified by MALDI-TOF mass spectrometry.
Results:
Nuclear extracts capable of transcriptional initiation and elongation and containing low amounts of cytoplasmic contaminations were prepared. 543 spots between 17 and 100 kDa and pI 3.7 and 8.8 have been resolved. From these, 65 spots were analyzed by MALDI-TOF mass spectrometry and 53 spots were identified as known proteins of which six represented transcription factors. Regulation by glucose was shown for the activator protein-1 component cJun. Since cJun was not visible on the silver stained 2-DE gel, western blotting of 1-DE gels and immunological detection had to be used in this case. The data were used to construct an online database.
Conclusions:
A 2-DE map and database of soluble nuclear proteins is presented. The identification of several transcription factors was possible on the silver-stained gels. However, further fractionation of the nuclear extracts will facilitate the detection of larger numbers of transcriptional regulators. The database and 2-DE map shown here may provide a useful reference for the identification of transcription factors from liver nuclei that are activated by different stimuli, e. g., nutrients.