Chemical Approaches for Quantification and Visualization of the Prenylome

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Mark Distefano from the University of Minnesota to develop methods to quantify and image the prenylome. Overall levels of prenylation will be measured by metabolic labeling with alkyne- or azide-containing substrate analogues followed by click reaction with a fluorescent reagent and subsequent quantification via flow cytometry. To selectively observe the localization of prenylated proteins such as K-Ras, alkyne- or azide-containing substrate analogues will be metabolically incorporated followed by click reaction with fluorescent probes and visualization via confocal microscopy. FRET-based methods and a strategy involving proximity-induced amplification will also be investigated to increase visualization sensitivity and selectivity. Results from the first objective will improve the understanding of how differences in prenylation levels correlate with various drug treatments and disease states. Results from the second objective will help clarify how the activities of prenylated proteins are regulated via changes in their subcellular localization.

Protein prenylation involves the attachment of a lipid group to a protein; several hundred of such proteins are present in human cells. Prenylated proteins are involved in regulating many features of cellular behavior including growth and communication and a number of diseases require the participation of such proteins. The focus of this proposal is to develop new methods to monitor the levels of prenylated proteins and track their location within cells. If this can be accomplished, new approaches to combat an array of diseases ranging from cancer to aging may emerge. This project will also be used as a vehicle to introduce a group of undergraduates to research by having them use the methods developed here to investigate protein prenylation in a wide variety of cell types and model organisms.