Sterol Carrier Protein-2 Protein Research Articles

In vitro and in silico studies of terpenes, terpenoids and related compounds with larvicidal and pupaecidal activity against Culex quinquefasciatus Say (Diptera: Culicidae)

BackgroundIn order to develop new larvicidal agents derived from phytochemicals, the larvicidal activity of fifty molecules that are constituent of essential oils was evaluated against Culex quinquefasciatus Say. Terpenes, terpenoids and phenylpropanoids molecules were included in the in vitro evaluation, and QSAR models using genetic algorithms were built to identify molecular and structural properties of biological interest. Further, to obtain structural details on the possible mechanism of action, selected compounds were submitted to docking studies on sterol carrier protein-2 (SCP-2) as possible target.ResultsResults showed high larvicidal activity of carvacrol and thymol on the third and fourth larval stage with a median lethal concentration (LC50) of 5.5 and 11.1 µg/mL respectively. Myrcene and carvacrol were highly toxic for pupae, with LC50 values of 31.8 and 53.2 µg/mL. Structure–activity models showed that the structural property π-bonds is the largest contributor of larvicidal activity while ketone groups should be avoided. Similarly, property–activity models attributed to the molecular descriptor LogP the most contribution to larvicidal activity, followed by the absolute total charge (Qtot) and molar refractivity (AMR). The models were statistically significant; thus the information contributes to the design of new larvicidal agents. Docking studies show that all molecules tested have the ability to interact with the SCP-2 protein, wherein α-humulene and β-caryophyllene were the compounds with higher binding energy.ConclusionsThe description of the molecular properties and the structural characteristics responsible for larvicidal activity of the tested compounds were used for the development of mathematical models of structure–activity relationship. The identification of molecular and structural descriptors, as well as studies of molecular docking on the SCP-2 protein, provide insight on the mechanism of action of the active molecules, and the information can be used for the design of new structures for synthesis as potential new larvicidal agents.

Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

NMR and X-ray structures of the putative sterol carrier protein 2 from Thermus thermophilus HB8 show conformational changes

Sterol carrier protein 2 (SCP-2), also known as nonspecific lipid transfer protein, is a ubiquitous intracellular ~13kDa protein found in mammals, insects, plants, archaea, and bacteria. Vertebrate SCP-2 has been implicated in a wide range of lipid-related functions in vitro, although its actual physiological role is still unknown. Tunnels in the protein serve as fatty acid binding vehicles. Here we report the first putative SCP-2 structure from a bacterium: specifically, the NMR and X-ray structures of the TTHA0401 protein (also designated as TT1886) from the extremely thermophilic bacterium Thermus thermophilus. The NMR structure and the two chain structures (chain A and chain B) of the asymmetric crystallographic unit (space group (P2(1)2(1)2(1))) revealed an internal cavity. However, this cavity is open to the outside, forming a tunnel, in only one of those structures (chain A, X-ray). The location of this tunnel is different from the one found in other SCP-2 proteins, and inaccessible cavities have not been seen before in SCP structures. We present evidence that at physiological concentrations, TTHA0401 likely exists as a monomer in equilibrium between open and closed conformations. This equilibrium is influenced by temperature-dependent dynamics, and is likely to be very different at the high temperatures preferred by this hyperthermophilic bacterium. Alternatively, another protein binding to TTHA0401 may induce a conformational change, which would constitute an intriguing metabolic regulation method in bacteria.

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.

A yeast sterol carrier protein with fatty-acid and fatty-acyl-CoA binding activity

The 14-kDa sterol carrier protein 2 (SCP2) domain is present in Eukaria, Bacteria and Archaea, and has been implicated in the transport and metabolism of lipids. We report the cloning, expression, purification and physicochemical characterization of a SCP2 from the yeast Yarrowia lipolytica (YLSCP2). Analytical size-exclusion chromatography, circular dichroism and fluorescence spectra, indicate that recombinant YLSCP2 is a well-folded monomer. Thermal unfolding experiments show that SCP2 maximal stability is at pH 7.0–9.0. YLSCP2 binds cis-parinaric acid and palmitoyl-CoA with K D values of 81 ± 40 nM and 73 ± 33 nM, respectively, sustaining for the first time the binding of fatty acids and their CoA esters to a nonanimal SCP2. The role of yeast SCP2 and other lipid binding proteins in transport, storage and peroxisomal oxidation of fatty acids is discussed.

Sterol carrier protein 2 gene transfer changes lipid metabolism and enterohepatic sterol circulation in mice

Background & Aims: Sterol carrier protein 2 (SCP-2) enhances sterol cycling and facilitates cholesterol translocation between intracellular organelles and plasma membrane in cultured cells, including hepatocytes. We examined the role of SCP-2 in hepatic cholesterol and lipid trafficking through the sinusoidal and canalicular secretory pathways of the liver in vivo. Methods: Recombinant adenovirus–mediated SCP-2 gene transfer was used to obtain hepatic overexpression of SCP-2 in C57BL/6 mice. Results: SCP-2 overexpression in the mouse liver resulted in an 8-fold increase of SCP-2 protein levels and determined various effects on lipid metabolism. It decreased high-density lipoprotein cholesterol and increased low-density lipoprotein (LDL) cholesterol concentrations. The expressions of hepatic LDL receptor, apolipoprotein (apo) A-I, apoB, and apoE were decreased. SCP-2 overexpression also increased hepatic cholesterol concentration, associated with decreased cholesterol neosynthesis. Increased biliary cholesterol and bile acid secretion, bile acid pool size, and intestinal cholesterol absorption were also observed. Conclusions: These results indicate that modulation of SCP-2 expression in the liver determines important modifications on lipoprotein metabolism, hepatic cholesterol synthesis and storage, biliary lipid secretion, bile acid metabolism, and intestinal cholesterol absorption.GASTROENTEROLOGY 2000;119:1708-1719

Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts

Although the sterol carrier protein 2 (SCP-2) gene encodes for two proteins, almost nothing is known of the function and potential processing of the larger transcript corresponding to the 58 kDa sterol carrier protein-2/3-oxo-acyl-CoA thiolase (SCP-x), in intact cells. L-cell fibroblasts transfected with cDNA encoding for the 58 kDa SCP-x protein had a 4.5-fold increase in SCP-x mRNA transcript levels. Western blot analysis showed SCP-x protein expression reached 0.011% of total protein, representing a 4.1-fold increase over basal levels. Surprisingly, the 13.2 kDa SCP-2 protein also increased 2-fold in the transfected cells. This was consistent with part of the 58 kDa SCP-x being proteolytically processed to 13.2 kDa SCP-2 as there was no evidence of an mRNA transcript corresponding to a 13.2/15.2 kDa gene product in the transfected L-cell clones. Confocal immunofluorescence microscopy of transfected L-cells showed that SCP-x/SCP-2 co-localized in highest concentration with catalase in peroxisomes, but significant amounts appeared extra-peroxisomal. Overexpression of SCP-x significantly altered cholesterol uptake and metabolism. Uptake of exogenous [3H]cholesterol and total cholesterol mass were increased 1.9- and 1.4-fold, respectively, in SCP-x expressors. Although cholesterol ester mass was unaltered, incorporation of exogenous [3H]cholesterol and [3H]oleic acid into cholesteryl esters increased 2.3- and 2.5-fold, respectively. These results from intact cells suggest the 13.2 kDa SCP-2 can arise from the larger SCP-2 gene product and indicate a role for the 58 kDa SCP-x protein in cholesterol uptake and intracellular cycling.—Atshaves, B. P., A. D. Petrescu, O. Starodub, J. B. Roths, A. B. Kier, and F. Schroeder. Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts. J. Lipid Res. 1999. 40: 610–622.

Reduced hepatic sterol carrier protein-2 expression in the streptozotocin treated diabetic rat

While a strong relationship between the hypercholesterolemia of diabetes and premature atherosclerosis is established, the etiology for the elevation in serum cholesterol in this disease is unknown. To determine whether diabetic hypercholesterolemia may be related to alterations in hepatic cholesterol transport capacity, sterol carrier protein-2 (SCP2) expression was examined in rats treated with streptozotocin (SZT). Furthermore, this study examined whether 17β-estradiol and insulin confer a protective effect on liver cholesterol homeostasis by maintaining hepatic SCP2 levels. SCP2 protein and mRNA expression were examined 13 days following SZT-induced diabetes onset and in diabetic rats treated with estradiol (1 cm silastic implant) or insulin (12 units/day). Data indicate that SCP2 protein levels were significantly reduced in the diabetic animals and that SCP2 protein expression in the liver was inversely related to the level of serum cholesterol in the diabetic animals. In contrast, SCP2 mRNA levels examined by slot blot, ribonuclease protection assay, and Northern blot analysis were significantly elevated. Both insulin and estradiol were able to enhance the expression of SCP2 protein in the liver following SZT treatment. The results of this investigation clearly indicate that hepatic SCP2 protein levels are significantly altered in the diabetic state suggesting that cholesterol transport capacity is reduced in the SZT-treated diabetic rat. The inverse relationship between serum cholesterol and hepatic SCP2 protein content suggests that the reduction in this protein may be a contributing factor in diabetic hypercholesterolemia.

Differential expression of hepatic sterol carrier proteins in the streptozotocin-treated diabetic rat.

Sterol carrier protein-2 (SCP2) is a 13.2-kilodalton protein that has been implicated in intracellular cholesterol transport, whereas a related sterol carrier protein, sterol carrier protein-X (SCPx; 58 kilodaltons) has been suggested to function also in the beta-oxidation of fatty acids. Although diabetes-related hyperlipidemia and altered cholesterol metabolism have been extensively studied, the intracellular cholesterol transport capacity during hyperglycemic states has not been examined. The fact that beta-oxidation is increased in diabetes whereas hepatic cholesterol metabolism is reduced suggests that differential expression of these sterol carrier proteins may accompany diabetic dyslipidemia. In this study, SCP2 protein levels were reduced by 60% in mildly hypercholesterolemic (cholesterol, > 130 and < 150 mg/dl; P < 0.01) diabetic rats and by 90% in severely hypercholesterolemic (cholesterol, > 150 mg/dl; P < 0.002) diabetic animals. In contrast, hepatic SCPx protein expression increased (3.5-fold) after diabetes induction with streptozotocin (STZ). The decline in SCP2 was inversely related to serum cholesterol levels. Hepatic SCP messenger RNA levels examined by ribonuclease protection assay demonstrated that hepatic SCP messenger RNA was increased 2-fold in diabetic animals. Northern blot analysis indicated that both the 0.8-kilobase SCP2-specific and the 2.1-kilobase SCPx-specific transcripts increased after STZ injection. SCPx protein induction preceded the decline in SCP2 by 4-5 days. Insulin treatment reversed the increase in SCPx and prevented the decline in SCP2. We conclude that SCP2 and SCPx are differentially expressed in the STZ-diabetic rat and suggest that this change in SCP expression should be considered a potential contributing mechanism through which cholesterol metabolism may be altered in diabetes.

Regulation of sterol carrier protein 2 (SCP2) gene expression in rat peritoneal macrophages during foam cell formation. A key role for free cholesterol content.

Sterol carrier protein 2 (SCP2) has been shown to be involved in intracellular transport and metabolism of cholesterol. However, there have been no reports concerning SCP2 in macrophages, the major source of atheromatous foam cells. We investigated whether SCP2 is present in rat peritoneal macrophages and determined the changes of SCP2 and its mRNA levels in macrophages during form cell formation induced by acetylated LDL (AcLDL). Immunoblot analysis and Northern blot analysis demonstrated that both SCP2 and its mRNA are expressed in rat peritoneal macrophages. Incubations with AcLDL caused a dose- and time-dependent increase of cellular esterified cholesterol, SCP2 and its mRNA in rat peritoneal macrophages. The inhibitor of acyl-CoA:cholesterol acyltransferase further enhanced AcLDL-induced increase of SCP2 protein and its mRNA. Incubations with 25-hydroxy cholesterol also caused a dose-dependent stimulation of SCP2 gene expression in macrophages, while incubation with maleylated BSA had no effect. These results suggest that the increment of cellular-free cholesterol is responsible for enhanced SCP2 gene expression in macrophages. The enhancement of SCP2 gene expression by AcLDL suggests that SCP2 may play an important role during foam cell formation induced by AcLDL which may be most important step for the atherosclerosis.

Regulation of sterol carrier protein-2 gene expression in rat liver and small intestine

Sterol carrier protein-2 (SCP2) is a peroxisomal protein most highly expressed in non-steroidogenic tissues such as liver and small intestine. We have examined SCP2 gene expression during development and after alterations in lipid and bile acid metabolism and compensatory cell growth in the rat. The developmental expression of SCP2 displayed a biphasic pattern of relative mRNA abundance with a peak at day 19 to 20 of fetal life, reaching adult levels by day 14 and after day 14 in small intestine. In adult rats there was no effect on SCP2 mRNA abundance, or the relative proportions of the four SCP2 transcripts after gemfibrozil treatment, 30-fold changes in hepatic cholesteryl ester and triglyceride levels, bile ligation, compensatory hepatic or renal growth. However, immunoblot analysis of tissue homogenates revealed that SCP2 protein is decreased by 75% in the livers of gemfibrozil-treated animals and increased by 5-fold at 48 h in regenerating liver and in the remaining kidney after unilateral nephrectomy. Taken together these results suggest that SCP2 gene expression is developmentally regulated and modulated translationally or post-translationally in the adult rat by gemfibrozil and compensatory cell growth.

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.

Cloning, expression, and nucleotide sequence of rat liver sterol carrier protein 2 cDNAs.

The structure and expression of rat sterol carrier protein 2 (SCP2) were studied by cloning and sequencing SCP2 cDNAs and by hybridizing the SCP2 cDNA to RNA from a variety of rat tissues. Initial screening of a rat liver cDNA library with an oligonucleotide probe derived from the rat SCP2 protein sequence revealed an 825-base pair cDNA clone coding for the complete SCP2 protein sequence. Our results suggest that rat liver SCP2 is 143 amino acid residues long, including a 20-amino acid residue prepeptide (pre-SCP2). Using the rat SCP2 cDNA as a probe for Northern blot hybridization analyses under high stringency conditions with rat liver poly(A) RNA, four mRNA species differing in their sizes and levels of expression are detected. The lengths of the corresponding mRNAs are 0.8, 1.4, 2.1, and 2.7 kilobases (kb), respectively. In all other tissues analyzed mRNAs hybridizing to the SCP2 cDNA probe were detected in levels much lower than in the liver. In these tissues the vast majority of the expressed SCP2 mRNAs consists of the 0.8-kb mRNA. Results from Southern blotting hybridization analyses with rat genomic DNA suggest that the four different mRNA species are products of a single gene. Extensive screening of rat liver cDNA libraries using rat liver SCP2 cDNA probes led to the isolation of 10 additional clones, all of them containing cDNA insertions longer than 0.8 kb. Restriction mapping and nucleotide sequencing analyses of these cDNAs indicate that eight extend upstream of the SCP2 cDNA, and two extend 5' as well as 3'. Translation of the sequence extending most upstream suggests that this cDNA is a nearly full-length cDNA coding for a protein of 547 amino acids (SCP chi). SCP chi contains a 404-amino acid amino-terminal extension as compared with pre-SCP2 whereas its carboxyl-terminal part is identical with pre-SCP2. Sequencing of the 3' extension cDNAs suggests that polyadenylation had occurred 651 nucleotides downstream as compared with the remaining cDNAs. A probe from this region hybridizes to the 1.4- and 2.7-kb mRNAs. From our data we conclude that the 1.4- and 2.7-kb mRNA species are generated by alternative polyadenylation of the 0.8- and 2.1-kb mRNAs, respectively. In contrast, the 2.1- and 2.7-kb mRNAs contain an approximately 1300-nucleotide extension upstream of the 0.8- and 1.4-kb mRNAs, suggesting differential splicing of the primary SCP2 gene transcript leading to expression of pre-SCP2 and SCP chi from a single gene.

Characterization of a cDNA Encoding Rat Sterol Carrier Protein2

Sterol carrier protein2 (SCP2) is a 13.2-kD protein that is thought to be involved in the intracellular transport of cholesterol. Using synthetic oligonucleotides based on the protein sequence of SCP2, a clone (SP43) was isolated from a rat liver cDNA library. The DNA sequence revealed that the cDNA could encode a polypeptide of 273 amino acids (28.9 kD) or 143 amino acids (15.3 kD) in which the carboxy-terminal 123 amino acids are identical to the SCP2 protein. RNA blot hybridization revealed that a variety of rat tissues contain a homologous RNA of a size similar to SP43 (approximately 1.5 kb). Levels of SCP2 mRNA increased in parallel with cytochrome P450scc mRNA in the immature gonadotropin-primed rat ovary. The isolation of a cDNA clone encoding SCP2 will facilitate studies on its role in cholesterol metabolism.