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PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions

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Sci. Signal.  29 Mar 2016:
Vol. 9, Issue 421, pp. rs2
DOI: 10.1126/scisignal.aad4337

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Detecting lipid-protein partners with PLIF

Phosphoinositides are a group of low-abundance lipid molecules in cellular membranes that regulate cellular physiology. Aberrant signaling in the pathways mediated by phosphoinositides underlies a wide range of diseases, including cancer, obesity, and neurodegenerative disorders. Ceccato et al. developed a method that enables fast and sensitive detection of phosphoinositide-binding partners than currently available approaches. The fluorescence-based method PLIF not only detected known phosphoinositide partners for various proteins but also determined relative binding affinities. The authors used their method to show that sorting nexins, which mediate vesicular trafficking, are promiscuous, interacting with many types of phosphoinositides. The high-throughput nature of PLIF makes it directly applicable to drug development aimed at disrupting 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.

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