Protein modification with hydrophobic prenyl groups in malignant cells

Sammanfattning: Mevalonate (MVA) is the key intermediate in the isoprene synthetic pathway leading to the formation of a number of biologically important lipid molecules. It is well known that inhibition of MVA synthesis blocks the cell cycle progression of mammalian cells, a fact suggesting that some isoprenoids derived from MVA are required for cell proliferation. For this reason the MVA pathway has been implicated in cancer. Among MVA products involved in cell proliferation, much attention has been paid at farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). FPP and GGPP constitute the isoprene substrates in prenylation of proteins (like Ras and nuclear lamins). FF or GG groups are linked to a cysteine (C) residue by a thioether bond at a CAAX (A, aliphatic amino acid and X, any amino acid) box of the carboxy terminal of the protein. A less common type of prenylation is di-geranylgeranylation at a CC or CXC box (Rab proteins). Since prenylation is crucial for the function of e.g. Ras and Rab, it has been proposed that protein prenylation may be important for malignant cell growth. The aim of this thesis was to characterize protein prenylation in human malignant cells regarding types of prenyl groups involved. We did not focus only on farnesylation and geranylgeranylation, but also on the possibility whether tumor cell proteins could be prenylated with long-chain isoprenes (dolichols). For this reason, we developed and optimized highly sensitive mass spectrometric methods to detect the prenyl groups. After labeling with radioactive dolichol (Dol) and detection of free Dol after cleavage of thioether bonds we could provide evidence that Dol is covalently linked to tumor cell proteins. Furthermore, after extensive enzymatic proteolysis of prenylated proteins we found a highly hydrophobic fraction (designated HPC), compared to the F cysteine (FC) and GG cysteine (GGC) fractions, containing Dol and cysteine. However, a closer analysis of HPC strongly suggested that it mainly represents di-geranylgeranylated CC or CXC peptide fragments. Notably, this type of prenylation was highly represented in cells of tumor origin compared to normal cells. In breast cancer and Ewing's sarcoma cells it was the predominating type of prenylation, and it was over represented in the tumor cell nuclei. Our results raise the possibility that di- geranylgeranylated proteins may be important for malignant cell growth.