The prenylation of proteins in mammalian cells is a recently discovered cellular event. Both 15-carbon farnesyl and 20-carbon geranylgeranyl groups are found at the C-termini of proteins such as ras proteins, heterotrimeric G proteins, GTP-binding proteins, and rhodopsin kinase. It has been shown that the prenylation of ras proteins is absolutely required for the transformation of cells expressing oncogenic forms of this protein. Thus, the study of the protein prenylation pathway may lead to better treatments for controlling human malignancies. A protein geranylgeranyltransferase that attaches geranylgeranyl groups to the cysteine residues near the C-terminal end of proteins using geranylgeranyl pyrophosphate as a substrate has been purified to homogeneity. The kinetic properties and substrate specificities of this enzyme will be further characterized. Inhibition of this enzyme as well as the inhibition of the protein farnesyltransferase by limonene and its oxidized metabolites will also be studied. Many prenylated proteins are further modified by proteolysis which removes the C-terminal three amino acids adjacent to the prenylated cysteine residue. This protease has been detected in cell membranes and continued work may lead to the purification of the relevant enzyme. Substrate specificity studies will be carried out with the protease in membranes or in purified form, if possible. Both H-ras and N-ras proteins are modified by farnesylation and palmitoylation. A protein palmitoyltransferase that acts on ras proteins has been partially purified. Efforts will be made to purify the enzyme to homogeneity so that it can be characterized. Attempts will also be made to clone the enzyme from a cDNA library and to express large amounts of this protein using the baculovirus/insect cell system. Protein prenyl groups probably serve as membrane anchors but very little is known about the function of these elements. The farnesyl group of ras is required for the activation of its downstream components in the cytosol of cells suggesting that the prenyl group may dictate protein-protein interactions. This possible role of protein prenyl groups will be explored by using ras proteins that are modified with farnesyl analogs. Such information may be the most useful for designing strategies for the treatment of ras-dependent tumors. Similar studies will be carried out with rhodopsin kinase.
Showing the most recent 10 out of 35 publications
