Avery L. McIntosh

Gene structure, intracellular localization, and functional roles of sterol carrier protein-2.

Since its discovery three decades ago, sterol carrier protein-2 (SCP-2) has remained a fascinating protein whose physiological function in lipid metabolism remains an enigma. Its multiple proposed functions arise from its complex gene structure, post-translational processing, intracellular localization, and ligand specificity. The SCP-2 gene has two initiation sites coding for proteins that share a common 13 kDa SCP-2 C-terminus: (1) One site codes for 58 kDa SCP-x which is partially post-translationally cleaved to 13 kDa SCP-2 and a 45 kDa protein. (2) A second site codes for 15 kDa pro-SCP-2 which is completely post-translationally cleaved to 13 kDa SCP-2. Very little is yet known regarding how the relative proportions of the two transcripts are regulated. Although all three proteins contain a C-terminal SKL peroxisomal targeting sequence, it is unclear why all three proteins are not exclusively localized in peroxisomes. However, the recent demonstration that the SCP-2 N-terminal presequence in pro-SCP-2 dramatically modulated the intracellular targeting coded by the C-terminal peroxisomal targeting sequence may account for the observation that as much as half of total SCP-2 is localized outside the peroxisome. The tertiary and secondary structure of the 13 kDa SCP-2, but not that of 15 kDa pro-SCP-2 and 58 kDa SCP-x, are now resolved. Increasing evidence suggests that the 58 kDa SCP-x and 45 kDa proteins are peroxisomal 3-ketoacyl-CoA-thiolases involved in the oxidation of branched chain fatty acids. Since 15 kDa pro-SCP-2 is post-translationally completely cleaved to 13 kDa SCP-2, relatively little attention has been focused on this protein. Finally, although the 13 kDa SCP-2 is the most studied of these proteins, because it exhibits diversity of its ligand partners (fatty acids, fatty acyl CoAs, cholesterol, phospholipids), new potential physiological function(s) are still being proposed and questions regarding potential compensation by other proteins with overlapping specificity are only beginning to be resolved.

Liver Fatty Acid Binding Protein and Obesity

While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity and metabolic syndrome. Consequently, mammals evolved fatty acid-binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15-member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as in intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age- and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair fed a high-fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age- or diet-induced obesity.

Liver Fatty Acid-binding Protein Targets Fatty Acids to the Nucleus REAL TIME CONFOCAL AND MULTIPHOTON FLUORESCENCE IMAGING IN LIVING CELLS

Although unesterified long chain fatty acids interact with peroxisome proliferator-activated receptors to initiate transcription within the nucleus, almost nothing is known regarding factors regulating long chain fatty acid distribution to the nucleus of living cells. The possibility that the liver fatty acid-binding protein (L-FABP) may function in this role was addressed in transfected L-cell fibroblasts overexpressing L-FABP using a series of fluorescent fatty acids differing in chain length and unsaturation. After 30 min of incubation, oxidation of BODIPY-, NBD-, and cis-parinaric acids was undetectable in L-cells. Likewise, L-cells very poorly esterified these fluorescent fatty acids in the following order: 0% BODIPY-C5, NBD-C6 (short chain length) < 0–3% NBD-C18, BODIPY-C16, cis-parinaric acid (long chain length) < 11% BODIPY-C12 (medium chain length). Real time confocal and multiphoton laser scanning microscopy (CLSM and MPLSM) showed that these fluorescent fatty acids were generally taken up in the following order: long chain (BODIPY-C16, NBD-C18) > medium chain (BODIPY-C12) ≫ short chain (BODIPY-C5, NBD-C6). The fluorescent fatty acids were imaged in the nucleus, primarily associated with the nuclear envelope, at levels about 2–3-fold lower than outside the nucleus. CLSM and MPLSM showed that L-FABP expression enhanced by 2–4-fold the initial rate and/or average maximal uptake of the long and medium chain but not the short chain fluorescent fatty acids in living cells. Furthermore, L-FABP expression increased the targeting of long and medium but not short chain fluorescent fatty acids to the nucleus by 2.9–4.4-fold and increased the proportion (i.e.nuclear:cytoplasm ratio) of medium and long chain but not short chain fatty acids by 2–3.6-fold. In summary, these results showed for the first time the presence of unesterified fatty acids in the nucleus of living cells and demonstrated that expression of a fatty acid-binding protein, L-FABP, specifically enhanced uptake and intracellular targeting of long and medium chain fatty acids to the nucleus.

Recent Advances in Membrane Microdomains: Rafts, Caveolae, and Intracellular Cholesterol Trafficking

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor/lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor/caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. Likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier protein-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomains in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of the lysosomal membrane, and/or intracellular cholesterol trafficking to select membranous organelles. Resolving these cholesterol pathways and the role of membrane cholesterol microdomains is essential to our understanding not only of processes that affect cholesterol metabolism, but also of the abnormal regulation that may lead to disease (diabetes, obesity, atherosclerosis, neutral lipid storage, Niemann-Pick C, congenital lipoid adrenal hyperplasia, etc.).

L-FABP directly interacts with PPARα in cultured primary hepatocytes

Although studies with liver type fatty acid binding protein (L-FABP) gene ablated mice demonstrate a physiological role for L-FABP in hepatic fatty acid metabolism, little is known about the mechanisms whereby L-FABP elicits these effects. Studies indicate that L-FABP may function to shuttle lipids to the nucleus, thereby increasing the availability of ligands of nuclear receptors, such as peroxisome proliferator-activated receptor-&agr; (PPAR&agr;). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPAR&agr;. L-FABP was shown to directly interact with PPAR&agr; in vitro through co-immunoprecipitation (co-IP) of pure proteins, altered circular dichroic (CD) spectra, and altered fluorescence spectra. In vitro fluorescence resonance energy transfer (FRET) between Cy3-labeled PPAR&agr; and Cy5-labeled L-FABP proteins showed that these proteins bound with high affinity (Kd approximately 156 nM) and in close proximity (intermolecular distance of 52Å). This interaction was further substantiated by co-IP of both proteins from liver homogenates of wild-type mice. Moreover, double immunogold electron microscopy and FRET confocal microscopy of cultured primary hepatocytes showed that L-FABP was in close proximity to PPAR&agr; (intermolecular distance 40–49Å) in vivo. Taken together, these studies were consistent with L-FABP regulating PPAR&agr; transcriptional activity in hepatocytes through direct interaction with PPAR&agr;. Our in vitro and imaging experiments demonstrate high affinity, structural molecular interaction of L-FABP with PPAR&agr; and suggest a functional role for L-FABP interaction with PPAR&agr; in long chain fatty acid (LCFA) metabolism.

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 [3H]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 1 2 near 1 min) and vesicular (t d 1 2 = 10–20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t b 1 2 decreased 36%, while the slower t d 1 2 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 1 2 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 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.

Liver fatty-acid-binding protein (L-FABP) gene ablation alters liver bile acid metabolism in male mice

Although the physiological roles of the individual bile acid synthetic enzymes have been extensively examined, relatively little is known regarding the function of intracellular bile acid-binding proteins. Male L-FABP (liver fatty-acid-binding protein) gene-ablated mice were used to determine a role for L-FABP, the major liver bile acid-binding protein, in bile acid and biliary cholesterol metabolism. First, in control-fed mice L-FABP gene ablation alone increased the total bile acid pool size by 1.5-fold, especially in gall-bladder and liver, but without altering the proportions of bile acid, cholesterol and phospholipid. Loss of liver L-FABP was more than compensated by up-regulation of: other liver cytosolic bile acid-binding proteins [GST (glutathione S-transferase), 3alpha-HSD (3alpha-hydroxysteroid dehydrogenase)], key hepatic bile acid synthetic enzymes [CYP7A1 (cholesterol 7alpha-hydroxylase) and CYP27A1 (sterol 27alpha-hydroxylase)], membrane bile acid translocases [canalicular BSEP (bile salt export pump), canalicular MRP2 (multidrug resistance associated protein 2), and basolateral/serosal OATP-1 (organic anion transporting polypeptide 1)], and positive alterations in nuclear receptors [more LXRalpha (liver X receptor alpha) and less SHP (short heterodimer partner)]. Secondly, L-FABP gene ablation reversed the cholesterol-responsiveness of bile acid metabolic parameters such that total bile acid pool size, especially in gall-bladder and liver, was reduced 4-fold, while the mass of biliary cholesterol increased 1.9-fold. The dramatically reduced bile acid levels in cholesterol-fed male L-FABP (-/-) mice were associated with reduced expression of: (i) liver cytosolic bile acid-binding proteins (L-FABP, GST and 3alpha-HSD), (ii) hepatic bile acid synthetic enzymes [CYP7A1, CYP27A1 and SCP-x (sterol carrier protein-x/3-ketoacyl-CoA thiolase)] concomitant with decreased positive nuclear receptor alterations (i.e. less LXRalpha and more SHP), and (iii) membrane bile acid transporters (BSEP, MRP2 and OATP-1). These are the first results suggesting a physiological role for the major cytosolic bile acid-binding protein (L-FABP) in influencing liver bile metabolic phenotype and gall-bladder bile lipids of male mice, especially in response to dietary cholesterol.

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 α-helix, 7-fold more β-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.

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of DHE crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric DHE with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems.