Protein prenylation is a critically important post-translational modification, and it has been estimated that several hundred different mammalian proteins are prenylated. The exact identity of all important prenylated proteins, and the extent of their prenylation, has not been established. This is now an issue of significant medical importance, due the potential use of prenylation inhibitors in treatment of human cancers. Moreover, there are indications that the statins, one of the most widely used drug classes, exert their therapeutic effects at least in part through the inhibition of protein prenylation. Recent clinical data has suggested that the statins serve as cancer chemopreventative agents, and cellular studies have indicated that this effect of the statins is due to their indirect inhibition of protein prenylation. Thus, the development of proteomic methods for the analysis, and particularly the quantitation of prenylated proteins, may be of major medical importance. The specific hypothesis of this proposal is that the cytostatic and apoptotic response of tumor cells to statins is due to the inhibition of the geranylgeranylation of signal transduction proteins in tumor cells. This collaborative proposal brings together the expertise of the Gibbs laboratory in the chemical and biochemical aspects of prenylation, with the bioanalytical and proteomic experience of the Regnier laboratory for the joint development and application of tools for the analysis of protein prenylation. The following specific aims are proposed: 1) To develop and evaluate new techniques for the physical isolation and subsequent characterization of prenylated proteins. A novel method developed in the Regnier laboratory, semipermeable surface (SPS) chromatography, will be investigated for its ability to selectively adsorb lipidated proteins. Subsequent desorption of the proteins will be followed by standard proteomic mass spectrometric analysis. 2) To develop and evaluate new techniques for the selective chemical modification, isolation and subsequent characterization of prenylated proteins. Several different chemical reactions selective for the unique olefin units present in the prenyl moieties will be evaluated for their ability to selectively derivatize prenylated peptides. The optimized versions of these methods will then be applied to the selective modification of prenylated proteins in a biological milieu. 3) To apply the techniques developed to the characterization of the prenylation status of proteins in cells treated with statins. These levels will be correlated with cytostatic, cytotoxJc, and apoptotic responses of pancreatic tumor cells. In parallel, the prenylation status of statin-treated cultured pancreatic beta-cells will also be investigated. It is critically important to develop biomarkers to validate the chemotherapeutic or chemopreventative response of patients to treatment with statins, farnesyltransferase inhibitors, and other drugs that may modulated protein prenylation. The proteomic analysis of this process would provide such a biomarker.
