Protein prenylation is a common post-translational modification present in eukaryotic cells. Many key proteins involved in signal transduction pathways are prenylated and inhibition of prenylation can be useful as a therapeutic intervention. While significant progress has been made in understanding protein prenylation in vitro, we have been interested in studying this process in living cells, including the question of where prenylated molecules localize. Here, we describe the synthesis and in vivo analysis of a series of fluorescently labeled multifunctional peptides, based on the C-terminus of the naturally prenylated protein CDC42. These peptides were shown to have intrinsic cell-penetrating abilities and enter cells through a passive transport mechanism and localize to the endomembrane surrounding the nucleus. Their cell-penetrating properties were shown to be mostly due to their prenylation state and not their peptide sequence. Once discovered other derivatives of these peptides were used to study peptide prenylation and enzymatic processing in living cells.
Also in this work other peptides and proteins were modified with non-natural prenyl diphosphates. This work aimed at honing in on the smallest alkyne or azide labeled prenyl diphosphate that is a substrate for PFTase. An alkyne containing PFTase substrate was identified that contained only 9 non-hydrogen atoms. The substrate was used to modify "Caax Box" containing proteins and peptides. Further proteolysis of the "Caax Box" allowed for alkyne modification of biomolecules with the addition of only a single modified cysteine. This result allows for the addition of alkyne functionality without the addition of a long hydrophobic chain that can hinder a biomolecules solubility or reactivity.
University of Minnesota Ph.D. dissertation. May 2009. Major: Chemistry. Advisor: Dr. Mark D. Distefano. 1 computer file (PDF); xviii, 156 pages.
Wollack, James W..
Exploitation of prenylation in biomolecules: cell-penetrating prenylated peptides and prenyalted proteins..
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