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.     

Ablation of the liver fatty acid binding protein gene decreases fatty acyl CoA binding capacity and alters fatty acyl CoA pool distribution in mouse liver

Although liver fatty acid binding protein (L-FABP) is known to bind not only long chain fatty acid (LCFA) but also long chain fatty acyl CoA (LCFA-CoA), the physiological significance of LCFA-CoA binding has been questioned and remains to be resolved. To address this issue, the effect of L-FABP gene ablation on liver cytosolic LCFA-CoA binding, LCFA-CoA pool size, LCFA-CoA esterification, and potential compensation by other intracellular LCFA-CoA binding proteins was examined. L-FABP gene ablation resulted not only in loss of L-FABP but also in concomitant upregulation of two other intracellular LCFA-CoA binding proteins, acyl CoA binding protein (ACBP) and sterol carrier protein-2 (SCP-2), by 45 and 80%, respectively. Nevertheless, the soluble fraction from livers of L-FABP (-/-) mice bound 95% less radioactive oleoyl-CoA than wild-type L-FABP (+/+) mice. The intracellular LCFA-CoA binding protein fraction (Fraction III) from wild-type L-FABP (+/+) mice, isolated by gel permeation chromatography of liver soluble proteins, exhibited one high-affinity binding and several low-affinity binding sites for cis-parinaroyl-CoA, a naturally occurring fluorescent LCFA-CoA. In contrast, high-affinity LCFA-CoA binding was absent from Fraction III of L-FABP (-/-) mice. While L-FABP gene ablation did not alter liver LCFA-CoA pool size, LCFA-CoA acyl chains of L-FABP (-/-) mouse livers were enriched 2.1-fold in C16:1 and decreased 1.9-fold in C20:0 fatty acids. Finally, L-FABP gene ablation selectively increased the amount of LCFAs esterified into liver phospholipid > cholesteryl ester, while concomitantly decreasing the amount of fatty acids esterified into triglycerides by 40%. In summary, these data with L-FABP (-/-) mice demonstrated for the first time that L-FABP is a physiologically significant contributor to determining liver cytosolic LCFA-CoA binding capacity, LCFA-CoA acyl chain distribution, and esterified fatty acid distribution.     

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.     

Role of regulatory F-domain in hepatocyte nuclear factor-4alpha ligand specificity

The F-domain of rat HNF-4alpha1 has a crucial impact on the ligand binding affinity, ligand specificity and secondary structure of HNF-4alpha. (i) Fluorescent binding assays indicate that wild-type, full-length HNF-4alpha (amino acids 1-455) has high affinity (Kd=0.06-12 nm) for long chain fatty acyl-CoAs (LCFA-CoA) and low affinity (Kd=58-296 nm) for unesterified long chain fatty acids (LCFAs). LCFA-CoA binding was due to close molecular interaction as shown by fluorescence resonance energy transfer (FRET) from full-length HNF-4alpha tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor), which yielded an intermolecular distance of 33 A, although no FRET to cis-parinaric acid was detected. (ii) Deleting the N-terminal A-D-domains, comprising the AF1 and DNA binding functions, only slightly affected affinities for LCFA-CoAs (Kd=0.9-4 nm) and LCFAs (Kd=93-581 nm). (iii) Further deletion of the F-domain robustly reduced affinities for LCFA-CoA and reversed ligand specificity (i.e. high affinity for LCFAs (Kd=1.5-32 nm) and low affinity for LCFA-CoAs (Kd=54-302 nm)). No FRET from HNF-4alpha-E (amino acids 132-370) tryptophan (FRET donor) to bound cis-parinaroyl-CoA (FRET acceptor) was detected, whereas an intermolecular distance of 28 A was calculated from FRET between HNF-4alpha-E and cis-parinaric acid. (iv) Circular dichroism showed that LCFA-CoA, but not LCFA, altered the secondary structure of HNF-4alpha only when the F-domain was present. (v) cis-Parinaric acid bound to HNF-4alpha with intact F-domain was readily displaceable by S-hexadecyl-CoA, a nonhydrolyzable thioether analogue of LCFA-CoAs. Truncation of the F-domain significantly decreased cis-parinaric acid displacement. Hence, the C-terminal F-domain of HNF-4alpha regulated ligand affinity, ligand specificity, and ligand-induced conformational change of HNF-4alpha. Thus, characteristics of F-domain-truncated mutants may not reflect the properties of full-length HNF-4alpha.     

Structure and function of the sterol carrier protein-2 N-terminal presequence

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2's affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.     

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.     

Pro-sterol carrier protein-2: role of the N-terminal presequence in structure, function, and peroxisomal targeting

Although the 20-amino acid presequence present in 15-kDa pro-sterol carrier protein-2 (pro-SCP-2, the precursor of the mature 13-kDa SCP-2) alters the function of SCP-2 in lipid metabolism, the molecular basis for this effect is unresolved. The presequence dramatically altered SCP-2 structure as determined by circular dichroism, mass spectroscopy, and antibody accessibility such that pro-SCP-2 had 3-fold less alpha-helix, 7-fold more beta-structure, 6-fold more reactive C terminus to carboxypeptidase A, 2-fold less binding of anti-SCP-2, and did not enhance sterol transfer from plasma membranes. These differences were not due to protein stability since (i) the same concentration of guanidine hydrochloride was required for 50% unfolding, and (ii) the ligand binding sites displayed the same high affinity (nanomolar K(d) values) in the order: cholesterol straight chain fatty acid > kinked chain fatty acid. Laser scanning confocal microscopy and double immunofluorescence demonstrated that pro-SCP-2 was more efficiently targeted to peroxisomes. Transfection of l-cells or McAR7777 hepatoma cells with cDNA encoding pro-SCP-2 resulted in 45% and 59% of SCP-2, respectively, colocalizing with the peroxisomal marker PMP70. In contrast, l-cells transfected with cDNA encoding SCP-2 exhibited 3-fold lower colocalization of SCP-2 with PMP70. In summary, the data suggest for the first time that the 20-amino acid presequence of pro-SCP-2 alters SCP-2 structure to facilitate peroxisomal targeting mediated by the C-terminal SKL peroxisomal targeting sequence.     

High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach

Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoprotein-mediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 A between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K(d) = 2 nM) at a single site. Thus, NBD-cholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to lipid droplets was correlated with the presence of adipose differentiation related protein, a lipid droplet-specific protein shown for the first time to bind unesterified sterol with high affinity.     

Lipid binding to sterol carrier protein-2 is inhibited by ethanol

Sterol carrier protein-2 (SCP-2) is an intracellular lipid carrier protein that binds cholesterol, phospholipids, fatty acids and other ligands. It has been reported that expression of SCP-2 was increased in brain nerve endings or synaptosomes of chronic ethanol-treated mice and it was shown that cholesterol homeostasis was altered in brain membranes of chronic ethanol-treated animals. Ethanol may interfere with the capacity of SCP-2 to bind cholesterol as well as other lipids. This hypothesis was tested using recombinant SCP-2 and fluorescent-labeled cholesterol, phosphatidylcholine (PC), and stearic acid. The association constants (K_a) of the ligand-SCP-2 complex were in the following order: NBD-cholesterol>NBD-PC>NBD-stearic acid. Ethanol, beginning at a concentration of 25 mM, significantly reduced the affinity of NBD-cholesterol and NBD-PC for SCP-2. Effects of ethanol on the K_a of NBD-stearic acid was significant only at the highest concentration that was examined (200 mM). Ethanol significantly increased the B_max of NBD-cholesterol for SCP-2 but did not have a significant effect on the B_max of NBD-PC. Similar results were found for effects of ethanol on the K_as and B_maxs using pyrene-labeled cholesterol and PC. In conclusion, ethanol beginning at a physiological concentration of 25 mM inhibited binding of cholesterol and PC to SCP-2. However, effects of ethanol on lipid binding to SCP-2 were dependent on the type of lipid. Ethanol in vivo may interfere with lipid binding to SCP-2 and disrupt lipid trafficking within cells.     

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.     

Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions

Sterol carrier protein 2 (SCP-2) participates in the microsomal conversion of lanosterol to cholesterol, in the conversion of cholesterol to cholesterol ester, and in intracellular cholesterol transfers. The stoichiometry of binding between cholesterol and SCP-2 is 1:1. However, reports have appeared attributing sterol carrier protein activity to a protein preparation identical to hepatic fatty acid-binding protein (FABP). Therefore, the present investigation was conducted to compare homogeneous preparations of FABP and SCP-2 with respect to their capacities to participate as carrier proteins in reactions involving sterols or fatty acids. The results show that SCP-2 and FABP have separate and distinct physiological functions, with SCP-2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP-2 does not specifically bind or transport fatty acid. As long as only small quantities of organic solvent (1.6 volume %) were used for substrate addition, the sterol delta 7-reductase liver microsomal assay for SCP-2 correlated well with the physiologically relevant assays employed in the reconstituted adrenal system. The sterol carrier protein activity previously attributed to rat hepatic FABP is explained by the presence of significant quantities of propylene glycol (15 volume %) or Tween 80 in the assay procedure.     

Decreased liver fatty acid binding capacity and altered liver lipid distribution in mice lacking the liver fatty acid-binding protein gene

Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.     

Expression of rat L-FABP in mouse fibroblasts: role in fat absorption

Fatty acid-binding proteins (FABP) are abundant cytosolic proteins whose level is responsive to nutritional, endocrine, and a variety of pathological states. Although FABPs have been investigatedin vitro for several decades, little is known of their physiological function. Liver L-FABP binds both fatty acids and cholesterol. Competitive binding analysis and molecular modeling studies of L-FABP indicate the presence of two ligand binding pockets that accomodate one fatty acid each. One fatty acid binding site is identical to the cholesterol binding site. To test whether these observations obtainedin vitro were physiologically relevant, the cDNA encoding L-FABP was transfected into L-cells, a cell line with very low endogenous FABP and sterol carrier proteins. Uptake of both ligands did not differ between control cells and low expression clones. In contrast, both fatty acid uptake and cholesterol uptake were stimulated in the high expression cells. In high expression cells, uptake of fluorescent cis-parinaric acid was enhanced more than that of trans-parinaric acid. This is consistent with the preferential binding of cis-fatty acids to L-FABP but in contrast to the preferential binding of trans-parinaric acid to the L-cell plasma membrane fatty acid transporter (PMFABP). These data show that the level of cytosolic fatty acids in intact cells can regulate both the extent and specificity of fatty acid uptake. Last, sphingomyelinase treatment of L-cells released cholesterol from the plasma membrane to the cytoplasm and stimulated microsomal acyl-CoA: cholesteryl acyl transferase (ACAT). This process was accelerated in high expression cells. These observations show for the first time in intact cells that L-FABP, a protein most prevalent in liver and intestine where much fat absorption takes place, may have a role in fatty acid and cholesterol absorption.Abbreviations FABP fatty acid-binding protein - L-FABP liver fatty acid-binding protein - I-FABP intestinal fatty acid-binding protein - H-FABP heart fatty acid-binding protein - A-FABP adipocyte fatty acid-binding protein - PMFABP plasma membrane fatty acid-binding protein - SCP-2 sterol carrier protein-2 - Dehydroergosterol (DHE) d-5,7,9(11),22-ergostatetraene-3b-ol - cis-parinaric acid-9Z, 11E, 13E, 15Z-octatetraenoic acid - trans parinaric acid, 9E, 11E, 13E, 14E-octatetraenoic acid - BSA bovine serum albumin - KRH Krebs-Ringer-Henseleit buffer     

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.     

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids, and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in Escherichia coli, purified to homogeneity by mutated streptavidin (SoftLink) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [(14)C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on- and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thereby validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) the ability to study lipids in their natural states (i.e., without relatively large reporter groups) and (ii) the ability to measure on- and off- rate constants as well as equilibrium constants.     

Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyl transferase I

Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.     

Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics

Although the most exogenous lipids enter the cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics, and response to lipid transfer proteins. SCP-2 expression decreased the mass of cholesterol and lyso-bis-phosphatidic acid [LBPA], as well as the ratios of cholesterol/phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5- and 3.8-fold, respectively, from lysosomal membranes isolated from SCP-2 expressing cells as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t(1/2)>4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells. These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore, the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane.