Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.

Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin.

Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic detail two competitively favorable membrane binding orientations of npc2 with a low interconversion barrier. The first binding mode (Prone) places the cholesterol binding pocket in direct contact with the membrane and is characterized by membrane insertion of a loop (V59-M60-G61-I62-P63-V64-P65). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required for strong membrane binding in Prone mode, and that it cannot be substituted by other anionic lipids. Meanwhile, sphingomyelin counteracts bmp by hindering Prone mode without affecting Supine mode. Our results provide concrete evidence that lipids modulate npc2-mediated cholesterol transport either by favoring or disfavoring Prone mode and that they impose this by modulating the accessibility of bmp for interacting with npc2. Overall, we provide a mechanism by which npc2-mediated cholesterol transport is controlled by the membrane composition and how npc2-lipid interactions can regulate the transport rate.

Bobbing of Oxysterols: Molecular Mechanism for Translocation of Tail-Oxidized Sterols through Biological Membranes.

Translocation of sterols between cellular membrane leaflets is of key importance in membrane organization, dynamics, and signaling. We present a novel translocation mechanism that differs in a unique manner from the established ones. The bobbing mechanism identified here is demonstrated for tail-oxidized sterols, but is expected to be viable for any molecule containing two polar centers at the opposite sides of the molecule. The mechanism renders translocation across a lipid membrane possible without a change in molecular orientation. For tail-oxidized sterols, the bobbing mechanism provides an exceptionally facile means to translocate these signaling molecules across membrane structures and may thus represent an important pathway in the course of their biological action.