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.

A facile, sensitive and quantitative membrane‐binding assay for proteins

“…Fitting the data to a one‐site binding isotherm revealed that GST‐2xP4M and GST‐PH showed 10‐fold higher apparent binding affinity ( K d ) for their cognate lipids (PI4P for GST‐2xP4M and PI(4,5)P 2 for GST‐PH) then for their noncognate lipids (Figure D). Note that our estimates of binding affinities are similar to those reported earlier using solution‐based liposome sedimentation assays but lower than those reported from assays where either the proteins or liposomes are surface‐immobilized (also see Ref. for a comparative review on binding affinities).…”

“…Similarly, we also tested binding of the pleckstrin homology (PH) domain of PLCδ1, a well‐known PI(4,5)P 2 biosensor . PLiMAP with GST‐PH domain revealed strongest crosslinks with BDPE on PI(4,5)P 2 ‐containing liposomes followed by PI(3,4,5)P 3 ‐ and PI(3,4)P 2 ‐containing liposomes (Figure A,C), and is similar in trend to that seen with the recently described single liposome binding assay . As an additional control, PLiMAP with glutathione S‐transferase (GST) alone showed no binding to any of the liposomes (Figure A).…”

Mapping Cell Membrane Organization and Dynamics Using Soft Nano-Imprint Lithography

“…7). This observation is consistent with the strong 21 and showed an absence of significant enrichment of PIs due to the pillar's topographical stimuli with an E ≤ 1.5 ( Supplementary Fig. 8a-b).…”

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

“…Fluorescent lipids, lipid-binding antibodies, or lipid biosensors are mainly used to study lipid localization (11,12). Most assays for studying lipid−protein interactions rely on reconstituted membranes/liposomes and are therefore largely restricted to soluble proteins (13)(14)(15)(16). The plethora of methods used to investigate these different processes makes it difficult to compare or validate their respective results.…”