The Bile Acid-Phospholipid Conjugate Ursodeoxycholyl-Lysophosphatidylethanolamide (UDCA-LPE) Disintegrates the Lipid Backbone of Raft Plasma Membrane Domains by the Removal of the Membrane Phospholipase A2

Wolfgang Stremmel,Gert Fricker,Ralf Weiskirchen,Simone Staffer

doi:10.3390/ijms20225631

Abstract

The bile acid-phospholipid conjugate ursodeoxycholyl-lysophosphatidylethanolamide (UDCA-LPE) was shown to have anti-inflammatory, antisteatotic, and antifibrotic properties, rendering it as a drug targeting non-alcoholic steatohepatitis (NASH). On a molecular level, it disrupted the heterotetrameric fatty acid uptake complex localized in detergent-resistant membrane domains of the plasma membrane (DRM-PM). However, its mode of action was unclear. Methodologically, UDCA-LPE was incubated with the liver tumor cell line HepG2 as well as their isolated DRM-PM and all other cellular membranes (non-DRM). The membrane cholesterol and phospholipids were quantified as well as the DRM-PM protein composition by Western blotting. The results show a loss of DRM-PM by UDCA-LPE (50 µM) with a 63.13 ± 7.14% reduction of phospholipids and an 81.94 ± 8.30% reduction of cholesterol in relation to mg total protein. The ratio of phospholipids to cholesterol changed from 2:1 to 4:1, resembling those of non-DRM fractions. Among the members of the fatty acid uptake complex, the calcium-independent membrane phospholipase A2 (iPLA2β) abandoned DRM-PM most rapidly. As a consequence, the other members of this transport system disappeared as well as the DRM-PM anchored fibrosis regulating proteins integrin β-1 and lysophospholipid receptor 1 (LPAR-1). It is concluded that UDCA-LPE executes its action by iPLA2β removal from DRM-PM and consequent dissolution of the raft lipid platform.

Highlights

Detergent resistant membrane domains within the plasma membrane (DRM-PM) are of importance for cell signaling, metabolic control, and cell to cell communication
It was shown to remove proteins from DRM-PM, i.e., the members of the fatty acid uptake complex, which is formed by caveolin-1, the membrane fatty acid binding protein (FABPPM), the cluster of differentiation (CD36) and the calcium-independent membrane phospholipase A2 known as PNPLA9 [2]
The finding of unaltered phospholipid content in the iPLA2β immunoprecipitate of the homogenate is in contrast to immunoprecipitation with flotillin-1, which is a key protein to establish DRM-PM microdomains involved in endocytosis, signal transduction, and cytoskeleton regulation [8]

Summary

Introduction

Detergent resistant membrane domains within the plasma membrane (DRM-PM) are of importance for cell signaling, metabolic control, and cell to cell communication. The question arises, whether the proteins are removed primarily or the structure of the DRM-PM, per se, is disrupted The latter is supported by the detergent-like character of the bipolar UDCA-LPE molecule, where the lipophilic LPE moiety could anchor within the phospholipid bilayer and solubilize the lipid structure. Another consideration is an extraction of lipids, i.e., cholesterol, as it is proposed for cyclodextrin [6,7]. This enzyme appears only to be indirectly involved in the fatty acid influx process. As a consequence of this structural interaction, iPLA2β could be removed together with bound phosphatidylcholine from the platform and the other proteins of the fatty acid uptake complex as well as other raft proteins stepwise fade from DRM-PM

Methods

Results

Discussion

Conclusion