Protein prenylation is a post-translational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions which contributes to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors (FTIs) based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in vivo. This dissertation describes our efforts to better understand the enzymology of protein prenylation in living systems. Initially, we prepared fluorescently labeled farnesylated peptides, based on the C-terminus of the naturally prenylated protein CDC42, to serve as substrates of the prenylation enzymes in living cells. These peptides are cell-permeable, can be imaged with confocal microscopy, quantified in cells with flow cytometry, and be detected by capillary electrophoresis after they have been processed by the cells own machinery. In addition to these peptides, we have also developed unnatural azide and alkyne containing isoprenoid moieties to serve as substrates for the prenyltransferase enzymes. Using the `click' reaction to a fluorophore we can quantify the levels of prenylated proteins in living systems and we show that this method is applicable to study the connection between protein prenylation and neurodegenerative disorders such as Alzheimer's disease. Taken together, our results highlight the applicability of these peptides and unnatural isoprenoid analogs as a platform for further study to better understand the enzymology of protein prenylation in living systems and to elucidate its role in certain diseases.
University of Minnesota Ph.D. dissertation. August 2012. Major: Chemistry. Advisor: Dr. Mark D. Distefano. 1 computer file (PDF); xv, 199 pages, appendices A-B.
Ochocki, Joshua Daniel.
Studies of protein prenylation in living systems: implications toward the better understanding of diseases.
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