title: PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions.
abstract: Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
authors.forename: Laurie
authors.initials: L
authors.lastname: Ceccato
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
authors.forename: Gaëtan
authors.initials: G
authors.lastname: Chicanne
authors.affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale (IPBS), 31000 Toulouse, France. Université de Toulouse, UPS (Université Paul Sabatier), IPBS, 31000 Toulouse, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Nahoum
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Pons
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France. CHU (Centre Hospitalier Universitaire) de Toulouse, Laboratoire d'Hématologie, 31059 Toulouse Cedex 03, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Payrastre
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Gaits-Iacovoni
authors.affiliation: INSERM, U1048 and Université Toulouse 3, I2MC, Avenue Jean Poulhès BP84225, 31432 Toulouse Cedex 04, France. julien.viaud@inserm.fr.
authors.forename: Julien
authors.initials: J
authors.lastname: Viaud
mesh_terms: D000818:Animals; D051379:Mice; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D058255:Protease Nexins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D018129:Phosphatidylinositol Phosphates; D058255:Protease Nexins; C055525:phosphatidylinositol 3-phosphate