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Updated: Dec 28, 2025
Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes
Published on: March 14, 2021
Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor
Tiphaine Péresse1, David Kovacs2, Mélody Subra2
1Institut de Chimie des Substances Naturelles, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France.
Abstract:
ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.
Insights
Schweinfurthin G (SWG) specifically targets oxysterol-binding protein (OSBP) and its lipid transfer activity. This fluorescent molecule acts as a tool to study OSBP function in cells and its role in lipid metabolism.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- ORPphilins, including schweinfurthins, are natural products that inhibit cancer cell growth.
- These compounds are proposed to target oxysterol-binding proteins (OSBP), which are crucial for lipid transfer between organelle membranes.
- The precise molecular mechanism of schweinfurthin action remains unclear.
Purpose of the Study:
- To elucidate the molecular mechanism by which schweinfurthin G (SWG) interacts with and inhibits OSBP.
- To characterize SWG as a pharmacological tool for studying OSBP function.
- To investigate the consequences of OSBP inhibition on cellular lipid distribution and trafficking.
Main Methods:
- Cellular sensitivity assays to determine the relationship between OSBP levels and SWG efficacy.
- Live-cell imaging using intrinsically fluorescent SWG to track its localization.
- In vitro membrane reconstitution assays to measure OSBP lipid transfer activity.
- Intermolecular Förster Resonance Energy Transfer (FRET) analysis to confirm direct binding.
Main Results:
- SWG's efficacy is inversely proportional to cellular OSBP levels.
- SWG specifically localizes to the trans-Golgi network in an OSBP-dependent manner.
- SWG directly binds to OSBP's lipid-binding cavity with nanomolar affinity, inhibiting its lipid transfer activity.
- Inhibition of OSBP by SWG disrupts post-Golgi trafficking, membrane cholesterol homeostasis, and PI(4)P turnover.
Conclusions:
- SWG is a specific, fluorescent inhibitor of OSBP lipid transfer activity.
- SWG serves as a valuable pharmacological tool for dissecting OSBP function at cellular and molecular levels.
- SWG binding to OSBP is sensitive to the membrane environment and inhibits the lipid exchange cycle.
