Abstract
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.