title: Metabolic Labeling with an Alkyne-modified Isoprenoid Analog Facilitates Imaging and Quantification of the Prenylome in Cells.
abstract: Protein prenylation is a post-translational modification that is responsible for membrane association and protein-protein interactions. The oncogenic protein Ras, which is prenylated, has been the subject of intense study in the past 20 years as a therapeutic target. Several studies have shown a correlation between neurodegenerative diseases including Alzheimer's disease and Parkinson's disease and protein prenylation. Here, a method for imaging and quantification of the prenylome using microscopy and flow cytometry is described. We show that metabolically incorporating an alkyne isoprenoid into mammalian cells, followed by a Cu(I)-catalyzed alkyne azide cycloaddition reaction to a fluorophore, allows for detection of prenylated proteins in several cell lines and that different cell types vary significantly in their levels of prenylated proteins. The addition of a prenyltransferase inhibitor or the precursors to the native isoprenoid substrates lowers the levels of labeled prenylated proteins. Finally, we demonstrate that there is a significantly higher (22%) level of prenylated proteins in a cellular model of compromised autophagy as compared to normal cells, supporting the hypothesis of a potential involvement of protein prenylation in abrogated autophagy. These results highlight the utility of total prenylome labeling for studies on the role of protein prenylation in various diseases including aging-related disorders.
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Charuta C
authors.initials: CC
authors.lastname: Palsuledesai
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Ochocki
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Kuhns
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Yen-Chih
authors.initials: YC
authors.lastname: Wang
authors.affiliation: Division of Environmental Health Sciences, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Janel K
authors.initials: JK
authors.lastname: Warmka
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Dustin S
authors.initials: DS
authors.lastname: Chernick
authors.affiliation: Division of Environmental Health Sciences, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Elizabeth V
authors.initials: EV
authors.lastname: Wattenberg
authors.affiliation: Department of Experimental and Clinical Pharmacology, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Arriaga
authors.affiliation: Department of Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001343:Autophagy; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D017368:Protein Prenylation; D013729:Terpenes / Q000737:chemistry*
chemical_list: D000480:Alkynes; D013729:Terpenes