Analysis of the prenylated proteome in living systems: Connecting dots between diseases and therapeutics

Abstract

Protein prenylation is a ubiquitous post-translational modification, involving attachment of an isoprenoid near the C-terminus of certain proteins. It plays a critical role in mediating membrane localization and protein-protein interactions. Inhibition of prenylation has been extensively studied for developing therapeutics against several types of cancers, and more recently against neurodegenerative disorders and parasitic diseases. However, their limited success in clinical trials indicates that much still remains unknown regarding the enzymology of protein prenylation. In this dissertation, our efforts to develop methods for quantification and identification of the prenylated proteome are described. We first demonstrate that the alkyne-functionalized isoprenoids metabolically label prenylated proteins, and allow for subsequent derivatization via “click” reaction. Next, we coupled metabolic labeling and differential gel electrophoresis to detect and identify proteins showing altered levels of prenylation in response to prenylation inhibitor. Additionally, we utilized metabolic labeling and the “click” reaction on intact cells to enable imaging and flow cytometry-based quantification of the prenylated proteome. Finally, we developed methods for profiling of prenylated proteome and detection of site of prenylation via mass spectrometry. Our results indicate that these methods should be applicable for elucidating the connection of protein prenylation and its inhibition with the neurodegenerative diseases and cancers.