The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis.

The ability of different lipid-binding proteins in liver cytosol to affect enzyme activities in bile-acid biosynthesis was studied in whole microsomes (microsomal fractions) and mitochondria and in purified enzyme systems. Sterol carrier protein2 stimulated the 7 alpha-hydroxylation of cholesterol and the 12 alpha-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol in microsomes and the 26-hydroxylation of cholesterol in mitochondria 2-3-fold. It also stimulated the oxidation of 5-cholestene-3 beta, 7 alpha-diol into 7 alpha-hydroxy-4-cholesten-3-one in microsomes. The stimulatory effect of sterol carrier protein2 was much less with purified cholesterol 7 alpha- and 26-hydroxylase systems than with microsomes and mitochondria. No stimulatory effect of sterol carrier protein2 was observed with purified 12 alpha-hydroxylase and 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase. Sterol carrier protein (fatty-acid-binding protein), 'DEAE-peak I protein' [Dempsey, McCoy, Baker, Dimitriadou-Vafiadou, Lorsbach & Howards (1981) J. Biol. Chem. 256, 1867-1873], ligandin (glutathione transferase B) and serum albumin had no marked stimulatory effects in either crude or in purified systems. The results suggest that sterol carrier protein2 facilitates the introduction of the less-polar substrates in bile-acid biosynthesis to the membrane-bound enzymes in crude systems in vitro. The broad substrate specificity appears, however, not to be consistent with a specific regulatory function for sterol carrier protein2 in bile-acid biosynthesis.     

Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2)

Chinese hamster ovary cells deficient in intact peroxisomes were compared with wild type cells for the presence of the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2). With the immunoblotting technique using the affinity purified antibody against rat liver nsL-TP, this protein was shown to be present in the homogenates from wild type cells, but could not be detected in mutant cells. In agreement with a previous study using livers from Zellweger patients it appears that there is a positive, as yet unknown, correlation between peroxisomes and the occurrence of nsL-TP in the cell. As a control using the affinity-purified antibody against the phosphatidylinositol transfer protein from bovine brain, levels of this protein were found to be normal in mutant cells. By use of metrizamide density gradients, nsL-TP was shown to cosediment with a membrane fraction different from peroxisomes. A protein of 58,000 daltons cross-reactive with the antibody against nsL-TP did cosediment with the peroxisomes in wild type cells and possibly with a "peroxisomal remnant" in the mutant cells. Incubation of wild type and mutant cells with L-[3-14C]serine showed that the biosynthesis of phosphatidylserine and the subsequent conversion into phosphatidylethanolamine was comparable in both cell types. This indicates that nsL-TP is not involved in the translocation of phosphatidylserine from the endoplasmic reticulum to the mitochondria as the site of decarboxylation.     

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.     

NMR structure of the sterol carrier protein-2: implications for the biological role

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone (15)N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded beta-sheet and four alpha-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the beta-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spin-labeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

The role of cyclic AMP in myogenesis.

The effect of exogenously administered cyclic AMP derivatives and of endogenously elevated cyclic AMP levels on the spontaneous fusion of skeletal muscle myoblasts has been investigated. Contrary to earlier reports, cAMP does not appear to have a direct inhibitory effect on the fusion of an established line (L8) of rat myoblasts. Similarly, cAMP did not block the fusion of primary chick myoblasts. However, fusion of the rat myoblasts was prevented when the cAMP induced inhibition of growth prevented the cells from reaching the "critical" cell density necessary for fusion.     

Interdomain interaction of cyclic AMP receptor protein in the absence of cyclic AMP

Interdomain interaction of apo-cyclic AMP receptor protein (apo-CRP) was qualified using its isolated domains. The cAMP-binding domain was prepared by a limited proteolysis, while the DNA-binding domain was constructed as a recombinant protein. Three different regions making interdomain contacts in apo-CRP were identified by a sequence-specific comparison of the HSQC spectra. The results indicated that apo-CRP possesses characteristic modules of interdomain interaction that are properly organized to suppress activity and to sense and transfer the cAMP binding signals. Particularly, the inertness of the DNA-binding motif in apo-CRP was attributable to the participation of F-helices in the interdomain contacts.     

Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x

Subcellular distribution of Aedes aegypti sterol carrier protein-2 (AeSCP-2) and AeSCP-x was studied using electron microscopy. In both cultured A. aegypti cells and in the larval midgut, AeSCP-2 was detected mostly in the cytosol, with some labeling mitochondria and nucleus, but not in membranous vesicles. The widespread distribution of AeSCP-2 in the midgut epithelium is consistent with its potential lipid transfer function in all phases of cholesterol absorption. In contrast, AeSCP-x was found mostly in the peroxisome. Differences in the subcellular distribution of AeSCP-2 and AeSCP-x suggest that these two members of the SCP-2 gene family are functionally distinct. Overexpression of AeSCP-2 in A. aegypti cells showed increased localization of AeSCP-2 to cytosol, mitochondria, and nucleus. This is the first report on the nuclear distribution of an SCP. Overexpression of AeSCP-2 resulted in increased cholesterol incorporation in cells, suggesting that AeSCP-2 enhances cholesterol uptake.     

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.     

cDNA sequence and bacterial expression of mouse liver sterol carrier protein-2.

Sterol carrier protein-2 (SCP-2) is an intracellular protein of Mr 13,096. In vitro studies have shown that it is involved in the transport and metabolism of cholesterol. This protein is believed to participate in these activities by forming a stoichiometric complex with the sterol. Because these activities occur in different intracellular locations, i.e. mitochondria, peroxisomes, and cytosol, it can be predicted that SCP-2 targets to these sites. In this report we show that a mouse cDNA (785 base pairs) encodes a precursor form of SCP-2 containing a N-terminal presequence and an additional C-terminal residue. These additional amino acid residues are found in proteins targeted to the mitochondria and peroxisomes, respectively. These signals are not found in SCP-2 purified from rat liver cytosol which is believed to be a cytosolic form. Northern analysis shows that there are four species of mRNA which hybridize to a SCP-2-specific probe at 1.0, 1.7, 2.2, and 2.9 kilobases. Southern analysis shows that the gene is distributed over a large amount of DNA or that there are multiple genes. We have cloned the cytosolic/peroxisomal form of mouse SCP-2 into the Escherichia coli expression vector pKK233-2 and have expressed and purified recombinant mouse SCP-2, Mr 13,034. The purified recombinant SCP-2 is immunoreactive to rabbit anti-rat SCP-2 antibody. It also has biological activity equivalent to homogeneous rat liver SCP-2 in stimulating the microsomal conversion of 7-dehydrocholesterol to cholesterol and in the esterification of cholesterol by acyl-CoA cholesterol acyltransferase by rat liver microsomes.