title: Design and application of in vivo FRET biosensors to identify protein prenylation and nanoclustering inhibitors.
abstract: Protein prenylation is required for membrane anchorage of small GTPases. Correct membrane targeting is essential for their biological activity. Signal output of the prenylated proto-oncogene Ras in addition critically depends on its organization into nanoscale proteolipid assemblies of the plasma membrane, so called nanoclusters. While protein prenylation is an established drug target, only a handful of nanoclustering inhibitors are known, partially due to the lack of appropriate assays to screen for such compounds. Here, we describe three cell-based high-throughput screening amenable Förster resonance energy transfer NANOclustering and Prenylation Sensors (NANOPS) that are specific for Ras, Rho, and Rab proteins. Rab-NANOPS provides the first evidence for nanoclustering of Rab proteins. Using NANOPS in a cell-based chemical screen, we now identify macrotetrolides, known ionophoric antibiotics, as submicromolar disruptors of Ras nanoclustering and MAPK signaling.
authors: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia.
authors: Monika
authors: M
authors: Köhnke
authors: Steven
authors: S
authors: Schmitt
authors: Nicholas
authors: N
authors: Ariotti
authors: Andrew M
authors: AM
authors: Piggott
authors: Robert G
authors: RG
authors: Parton
authors: Ernest
authors: E
authors: Lacey
authors: Robert J
authors: RJ
authors: Capon
authors: Kirill
authors: K
authors: Alexandrov
authors: Daniel
authors: D
authors: Abankwa
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D017368:Protein Prenylation / Q000187:drug effects*; D000090063:Proto-Oncogene Mas; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D020691:rab GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D020691:rab GTP-Binding Proteins; D018631:ras Proteins; D020741:rho GTP-Binding Proteins