Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.     

Sterol carrier protein-2 selectively alters lipid composition and cholesterol dynamics of caveolae/lipid raft vs nonraft domains in L-cell fibroblast plasma membranes

Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.     

Regulation of sterol transport between membranes and NPC2

Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.     

Sterol transfer between cyclodextrin and membranes: similar but not identical mechanism to NPC2-mediated cholesterol transfer

Niemann--Pick C disease is an inherited disorder in which cholesterol and other lipids accumulate in the late endosomal/lysosomal compartment. Recently, cyclodextrins (CD) have been shown to reduce symptoms and extend lifespan in animal models of the disease. In the present studies we examined the mechanism of sterol transport by CD using in vitro model systems and fluorescence spectroscopy and NPC2-deficient fibroblasts. We demonstrate that cholesterol transport from the lysosomal cholesterol-binding protein NPC2 to CD occurs via aqueous diffusional transfer and is very slow; the rate-limiting step appears to be dissociation of cholesterol from NPC2, suggesting that specific interactions between NPC2 and CD do not occur. In contrast, the transfer rate of the fluorescent cholesterol analogue dehydroergosterol (DHE) from CD to phospholipid membranes is very rapid and is directly proportional to the acceptor membrane concentration, as is DHE transfer from membranes to CD. Moreover, CD dramatically increases the rate of sterol transfer between membranes, with rates that can approach those mediated by NPC2. The results suggest that sterol transfer from CD to membranes occurs by a collisional transfer mechanism involving direct interaction of CD with membranes, similar to that shown previously for NPC2. For CD, however, absolute rates are slower compared to NPC2 for a given concentration, and the lysosomal phospholipid lysobisphosphatidic acid (LBPA) does not stimulate rates of sterol transfer between membranes and CD. As expected from the apparent absence of interaction between CD and NPC2, the addition of CD to NPC2-deficient fibroblasts rapidly rescued the cholesterol accumulation phenotype. Thus, the recent observations of CD efficacy in mouse models of NPC disease are likely the result of CD enhancement of cholesterol transport between membranes, with rapid sterol transfer occurring during CD--membrane interactions.     

Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.     

Sterol carrier protein-2 expression modulates protein and lipid composition of lipid droplets

Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.     

Sterol carrier protein-2 expression alters phospholipid content and fatty acyl composition in L-cell fibroblasts

The effects sterol carrier protein-2 (SCP-2) expression on L-cell phospholipid levels and fatty acyl composition was assessed using L-cells transfected with the murine cDNA encoding for either the 15 kDa proSCP-2 or 13.2 kDa SCP-2. Expression of these proteins reduced total phospholipid mass (nmol/mg protein) by 24% and reduced the cholesterol to phospholipid ratio 60 and 28%, respectively. In 15 kDa proSCP-2 expressing cells, individual phospholipid class masses, excluding sphingomyelin (CerPCho), were reduced as follows: phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) > ethanolamine glycerophospholipid (EtnGpl) > choline glycerophospholipid (ChoGpl). Furthermore, ethanolamine plasmalogen mass was decreased 25%, while choline plasmalogen mass was elevated 30% in 15 kDa proSCP-2 expressing cells. In 13.2 kDa SCP-2 expressing cells, phospholipid class mass was decreased as follows: PtdIns and PtdSer > ChoGpl. These changes in phospholipid mass resulted in altered cellular phospholipid composition. Expression of either protein differentially altered the type of fatty acid esterified onto the phospholipids. These effects included a greater proportion of polyunsaturated fatty acids and a reduction in saturated fatty acids, although 15 kDa proSCP-2 expression had a more robust effect on these parameters than did 13.2 kDa SCP-2 expression. In summary, expression of SCP-2 reduced individual phospholipid class mass, except for CerPCho, and altered the fatty acid composition of each phospholipid class examined.These results clearly demonstrate that SCP-2 expression altered basal phospholipid levels, suggesting that SCP-2 can alter the function of endoplasmic reticulum phospholipid synthetic enzymes.     

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.     

Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.     

Sterol carrier protein-2 suppresses microsomal acyl-CoA hydrolysis

Although sterol carrier protein 2 (SCP-2) has long been regarded primarily as a sterol transfer protein, its actual physiological function is not known. The recent discovery that SCP-2 binds long chain fatty acyl-CoAs (LCFA-CoAs) with high affinity suggests additional roles for SCP-2 in cellular utilization of LCFA-CoAs for synthesis of glycerides and cholesterol esters. Concomitant to these anabolic pathways, LCFA-CoAs are also degraded by cellular hydrolases. The purpose of the work presented herein was to determine if SCP-2 altered the aqueous pool of LCFA-CoA by (i) extracting LCFA-CoA from microsomal membranes, and (ii) protecting LCFA-CoA from microsomal hydrolase activity. The data demonstrated for the first time that SCP-2 increases the aqueous pool of oleoyl-CoA by increasing the aqueous/membrane distribution oleoyl-CoA by 2.4-fold. In addition, SCP-2 inhibited the hydrolysis of oleoyl-CoA by microsomal acyl-CoA hydrolase 1.6-2.4 fold, depending on the concentration of oleoyl-CoA. By simultaneously extracting LCFA-CoA from membranes and inhibiting LCFA-CoA degradation SCP-2 may potentiate LCFA-CoA transacylation and modulate the role of LCFA-CoAs as intracellular signaling molecules.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Intracellular sterol distribution in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

The potential role of liver fatty acid binding protein (L-FABP) in modulating cellular sterol distribution was examined in mouse L-cell fibroblasts transfected with cDNA encoding L-FABP. L-cells were chosen because they contain only a small amount of endogenous FABP which does not bind [3H]cholesterol, does not enhance intermembrane sterol transfer, and whose content is unaltered by the expression of L-FABP. Transfected L-cells expressed 0.34% of cytosolic protein as L-FABP. Transfection alone with low expression of L-FABP (0.008% of cytosolic protein) had no effect on any of the parameters tested. Three aspects of cellular sterol transfer were examined. First, cellular sterol uptake, monitored by [3H]cholesterol and the fluorescent sterol, delta-5,7,9(11),22-ergostatetraen-3 beta-ol, was increased 21.5 +/- 2.6% (p less than 0.001) in L-cells expressing L-FABP. This increase was not accounted for by increased sterol esterification in the cells expressing L-FABP. Inhibition of both cholesterol transfer and esterification with 3-(decyldimethylsilyl)-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide from Sandoz abolished the L-FABP related enhancement of both [3H]cholesterol uptake and esterification. Second, plasma membrane transbilayer distribution of sterol, determined by fluorescence methods indicated that the majority of sterol was in the inner leaflet of the plasma membrane. In transfected cells expressing L-FABP, twice as much sterol (28 +/- 4%) was present in the exofacial leaflet of the plasma membrane as compared to that of control cells (15 +/- 2%). Third, expression of L-FABP enhanced sterol transfer from the plasma membrane to microsomes in intact cells. Treatment of [3H]cholesterol or [3H]oleate-loaded cells with sphingomyelinase resulted in increased formation of radiolabeled cholesterol ester, consistent with enhanced microsomal esterification of plasma membrane derived cholesterol. Concomitantly, plasma membrane [3H]cholesterol became less accessible to oxidation by cholesterol oxidase. Sphingomyelinase-stimulated cholesterol esterification was 21 +/- 3% greater in transfected cells. Concomitantly, accessibility of plasma membrane [3H]cholesterol to cholesterol oxidase was decreased 18 +/- 3% in cells expressing L-FABP. These differences are consistent with the ability of L-FABP to influence sterol transport and plasma membrane transbilayer sterol distribution in intact cells.     

Membrane cholesterol dynamics: cholesterol domains and kinetic pools

Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes.     

Effects of Chronic Ethanol Consumption on Sterol Transfer Proteins in Mouse Brain

Abstract: Although lipids are essential to brain function, almost nothing is known of lipid transfer proteins in the brain. Early reports indicates cross-reactivity of brain proteins with antisera against two native liver sterol transfer proteins, sterol carrier protein-2 (SCP-2) and the liver form of fatty acid-binding protein (L-FABP). Herein, polyclonal antibodies raised against the recombinant liver sterol transfer proteins SCP-2 and L-FABP were used to identify the lipid transfer proteins in the brains of alcohol-treated and control mice. L-FABP was not detectable in brain of either control or chronic ethanol-treated mice. In contrast, SCP-2 not only was present, but its level was significantly ( p < 0.05) increased 23 and 50%, respectively, in brain homogenates and synaptosomes of mice exposed to alcohol. To determine whether antibodies against the recombinant liver SCP-2 reflected true levels of SCP-2 in brain, the cDNA sequence for brain SCP-2 was isolated from a brain cDNA library. The mouse brain SCP-2 sequence was 99.99% identical to the mouse liver SCP-2 sequence. The translated sequence differed by only one amino acid, and the replacement was conservative. Thus, unlike the fatty acid binding proteins, the SCP-2 moieties of brain and liver are essentially identical. Polyclonal antibodies against acyl-CoA binding protein, a lipid-binding protein that does not bind or transfer sterol, showed that increased levels of brain SCP-2 with chronic ethanol consumption did not represent a general increase in content of all lipid transfer proteins. Changes in the amount of SCP-2 may contribute to membrane tolerance to ethanol.     

Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2

The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.     

Sterol carrier protein-2 functions in phosphatidylinositol transfer and signaling

Over 20 years ago, it was reported that liver cytosol contains at least two distinct proteins that transfer phosphatidylinositol in vitro, phosphatidylinositol transfer protein (PITP) and a pH 5.1 supernatant fraction containing sterol carrier protein-2 (SCP-2). In contrast to PITP, there has been minimal progress on the structural and functional significance of SCP-2 in phosphatidylinositol transport. As shown herein, highly purified, recombinant SCP-2 stimulated up to 13-fold the rapid (s) transfer of radiolabeled phosphatidylinositol (PI) from microsomal donor membranes to highly curved acceptor membranes. SCP-2 bound to microsomes in vitro and overexpression of SCP-2 in transfected L-cells resulted in the following: (i) redistribution of phosphatidylinositols from intracellular membranes (mitochondria and microsomes) to the plasma membrane; (ii) enhancement of insulin-mediated inositol-triphosphate production; and (iii) 5.5-fold down regulation of PITP. Like PITP, SCP-2 binds two ligands required for vesicle budding from the Golgi, PI, and fatty acyl CoA. Double immunolabeling confocal microscopy showed SCP-2 significantly colocalized with caveolin-1 in the cytoplasm (punctate) and plasma membrane of SCP-2 overexpressing hepatoma cells (72%), HT-29 cells (58%), and SCP-2 overexpressing L-cells (37%). Taken together, these data show for the first time that SCP-2 plays a hitherto unrecognized role in intracellular phosphatidylinositol transfer, distribution, and signaling.