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Sterol carrier protein-2
Biochimica et Biophysica Acta 1486 (2000) 45^54 www.elsevier.com/locate/bba
Review
Sterol carrier protein-2 Udo Seedorf *, Peter Ellinghaus, Jerzy Roch Nofer Institute for Arteriosclerosis Research, Institute for Clinical Chemistry and Laboratory Medicine, Interdisciplinary Center for Clinical Research, Westphalian Wilhelms-University, D-48149 Mu«nster, Germany Received 6 October 1999; received in revised form 9 November 1999; accepted 9 November 1999
Abstract The compartmentalization of cholesterol metabolism implies target-specific cholesterol trafficking between the endoplasmic reticulum, plasma membrane, lysosomes, mitochondria and peroxisomes. One hypothesis has been that sterol carrier protein-2 (SCP2, also known as the non-specific lipid transfer protein) acts in cholesterol transport through the cytoplasm. Recent studies employing gene targeting in mice showed, however, that mice lacking SCP2 and the related putative sterol carrier known as SCPx, develop a defect in peroxisomal L-oxidation. In addition, diminished peroxisomal K-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in these null mice was attributed to the absence of SCP2 which has a number of properties supporting a function as carrier for fatty acyl-CoAs rather than for sterols. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Sterol carrier protein-2 ; Cholesterol; Peroxisome; L-Oxidation; Acyl-coenzyme A
1. Intracellular cholesterol tra¤cking ^ the sterol carrier hypothesis In most eukaryotic cells, the bulk of cholesterol is synthesized at the endoplasmic reticulum (ER) whereas almost 90% of the free, non-esteri¢ed fraction of this essential membrane lipid resides in the plasma membrane [1]. Cholesterol is found mainly at
Abbreviations: ACO, acyl-CoA oxidase; ACTH, adrenocorticotrophic hormone; ER, endoplasmic reticulum; LH, luteinizing hormone; NPC, Niemann-Pick disease type C; PBE, peroxisomal bifunctional enzyme; PPARK, peroxisome proliferator-activated receptor-K; SCP, sterol carrier protein; StAR, steroidogenic acute regulatory protein; VLCFA, very long chain fatty acid; X-ALD, X-linked adrenoleukodystrophy * Corresponding author. E-mail: [email protected]
the inner lea£et of the bilayer, where it limits membrane £uidity which is thought to stabilize the complex supramolecular structures that are formed between lipids, receptors, adaptor proteins and the cytoskeleton at the cell surface. The cholesterol hydroxyl group forms a hydrogen bond with a phospholipid carbonyl oxygen atom whereas the bulky steroid moiety and the £exible hydrocarbon tail are directed to the hydrophobic inner portion of the membrane. It was proposed that cholesterol is not evenly distributed within the inner lea£et of the membrane, but that it is concentrated in cholesterol-rich micro-domains called caveolae [2,3]. Caveolae are rich in sphingomyelin and VIP21 caveolin, a 21^ 24 kDa integral membrane protein that binds cholesterol at a 1:1 molar ratio [2,4^7]. The highly asymmetric distribution of cholesterol in cells makes it conceivable that intracellular tra¤cking of cholesterol requires target-speci¢c transport mechanisms that
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mediate its translocation from the site of synthesis at the ER to the caveolae of the plasma membrane. Moreover, one must postulate means that keep cholesterol from di¡using to the bilayer's outer lea£et and that inhibit its lateral di¡usion within the membrane. At present, not much is known about the precise mechanisms that establish the remarkable speci¢city of the machinery that regulates these fundamental processes in the cell. Speci¢c transport mechanisms have been made responsible for the transfer of cholesterol from the membranes of secondary lysosomes to the endoplasmic reticulum, the intracellular site of cholesterol esteri¢cation catalyzed by acyl-CoA cholesterol acyl transferase. A breakthrough discovery came with the identi¢cation of the genetic basis underlying Niemann-Pick disease type C (NPC), mapped to human chromosome 18 [8,9]. NPC is an inherited lipid storage disorder that a¡ects the viscera and central nervous system. Defective cholesterol tra¤cking leading to lysosomal accumulation of low density lipoprotein-derived free cholesterol is a characteristic feature of NPC cells. The mutated protein, which was named NPC1, shows homologies with the sterol sensing domains which are present in several key regulators of cholesterol homeostasis and a class of morphogen receptors called sonic hedgehog proteins (shh). NPC1 functions most likely in regulating the retroendocytic tra¤cking of cholesterol and other lysosomal cargo. Another important transport route of cholesterol, which is required mainly for the synthesis of pregnenolone and bile acids, concerns the delivery of sterols to mitochondria. Stimulation of steroid-producing cells of the gonads and adrenals with the trophic hormones luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) leads to a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the ¢rst committed step in steroid synthesis is performed by the side chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. One protein that is known to play a crucial role in this cholesterol translocation step was named steroidogenic acute regulatory protein (StAR) [10]. Lack of functional StAR causes the autosomal recessive disease
congenital lipoid adrenal hyperplasia, which is characterized by markedly impaired gonadal and adrenal steroid hormone synthesis [10]. StAR protein is expressed almost exclusively in steroid-producing cells and its presence is correlated with steroid hormone production. More recently, it could be shown that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation [11]. Little is known at present about sterol transport through the cytoplasm. Earlier studies supported that the bulk £ow of de novo synthesized cholesterol from the ER to the plasma membrane is a fast, energy-requiring process that proceeds independently of the secretory protein pathway, but may nevertheless be vesicular [12]. Alternatively, cholesterol transport through the cytoplasm could be achieved via soluble carrier proteins that would shield the lipophilic transport substrate from the aqueous phase and harbor signals that could mediate target specificity. Unfortunately, e¡orts that were aimed at isolating such proteins and cloning their corresponding cDNAs have essentially been unsuccessful. The best studied candidate for a soluble sterol carrier has been sterol carrier protein-2 (SCP2), also known as the non-speci¢c lipid transfer protein, puri¢ed almost 20 years ago on the basis of its ability to activate the enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes in vitro [13]. Because SCP2 promotes the exchange of a wide variety of sterols between membranes in vitro and its expression a¡ects sterol tra¤cking in certain tissue culture systems, it has long been thought that the protein acts as a substrate carrier in various steps of sterol metabolism [14^16]. On the other hand, low sterol transfer activity under physiologic conditions, apparent lack of speci¢city for cholesterol, and the predominant localization of SCP2 in peroxisomes made it di¤cult to understand how the protein might carry out this role in the intact cell. The purpose of this review is to summarize our current knowledge about the molecular biology of the sterol carrier protein-2 gene family and discuss potential functions of SCP2 in vivo, as studied in a murine model of complete SCP2 de¢ciency generated by gene targeting [17].
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2. The SCP2 gene family At present, the SCP2 gene family includes only four distinct members (SCP2, SCPx, D-PBE and UNC-24/hSLP-1), but it can be expected that additional homologues may be identi¢ed in the future. Apart from SCP2, which is expressed as an individual protein, the other homologues contain their SCP2 domains at the C-terminus (Fig. 1). Mammalian SCP2 is synthesized as a 143 amino acid precursor that is processed most likely in peroxisomes to the 123 amino acid mature SCP2. The human SCP2encoding gene comprises 16 exons, which span approx. 100 kb on chromosome 1p32 [18^20]. Alternate transcription initiation regulates the expression of SCP2 and a second gene product that consists of 547 amino acids (named sterol carrier protein-x, SCPx) [21]. SCPx represents a fused protein consisting of a thiolase, extending from amino acid 1 to 404, and SCP2 which is located at the carboxyl terminus [22,23]. The fused gene can be traced back to Drosophila melanogaster [24] (GenBank accession No. X97685), whereas two separated genes for SCP2 and the thiolase are present in Caenorhabditis elegans and several yeast species [25,26]. An ancient precursor of the SCP2 sequence could be identi¢ed even in the methanogenic archaeon, Methanococcus jannaschii [27]. It is known from in vitro studies that SCPx has lipid transfer activity similar to SCP2 [28]. The substrate speci¢city of the SCPx thiolase shows a preference for medium straight-chain acyl-CoA substrates, 2-methyl-branched-chain fatty acyl-CoAs (such as 3-ketopristanoyl-CoA) and bile acid precursors (such as 3K,7K,12K-trihydroxy-24-ketocholestanoyl-CoA) [29^31]. The last two substrates are not oxidized e¡ectively in mitochondria but require peroxisomal L-oxidation, which coincides with the peroxisomal localization of SCPx. The properties of the newly discovered SCPx thiolase thus di¡er clearly from the long-known peroxisomal thiolase, that was identi¢ed by Hashimoto and coworkers almost 15 years ago [32]. Unlike SCPx, this enzyme acts preferentially on the 3-keto derivatives of the straight very long chain fatty acid (VLCFA) that are metabolized in peroxisomal L-oxidation [32,33]. An interesting ¢nding was that the cDNA encoding an 80 kDa precursor cloned originally as 17L-
Fig. 1. Domain structure of the currently known members of the SCP2 gene family. Amino acid numbers of known processing sites are indicated by arrows. SCP2, sterol carrier protein-2 ; SCPx, sterol carrier protein-x; PBE, peroxisomal bifunctional enzyme; hSLP, human stomatin-like protein.
hydroxysteroid dehydrogenase type IV (17L-HSD4) contained a C-terminal domain similar to SCP2 [34]. The domain was fused to a peptide with acylCoA 2-enoyl hydratase/3-hydroxyacyl-CoA dehydrogenase activity (also known as peroxisomal bifunctional enzyme (PBE)) [35]. Processing occurs after import of the 80 kDa precursor into peroxisomes at the junction between the acyl-CoA 2-enoyl hydratase and 3-hydroxyacyl-CoA dehydrogenase domains [36]. The SCP2-like domain is required for import of the 80 kDa precursor into peroxisomes and confers a similar intrinsic lipid transfer activity to the fusion protein as was demonstrated for SCPx [37]. In contrast to the well known PBE enzyme that is also called L-bifunctional enzyme [38], because it converts trans-enoyl-CoA to their 3-keto derivatives via the L-hydroxy stereoisomer, the new PBE enzyme catalyzes the same transformation via the D-stereoisomer [39,40]. Therefore, Hashimoto and coworkers have introduced the name `D-bifunctional enzyme' for the peroxisomal protein that was originally cloned as 17L-HSD4. The two PBE proteins di¡er in their substrate speci¢city. Whereas the D-PBE catalyzes the formation of 3-ketoacyl-CoA intermediates from straight and 2-methyl-branched-chain fatty acyl-CoAs, the activity of L-PBE is only high with the straight-chain substrates [39^42]. Convincing evidence showing that these ¢ndings are relevant also in vivo were obtained by van Grunsven et al. who identi¢ed a patient with isolated D-PBE de¢ciency [43]. Besides a severe block in pristanic acid L-oxidation,
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Fig. 2. Schematic illustration of cellular phytanic acid tra¤cking and metabolism. L-FABP, liver fatty acid binding protein ; SCP2, sterol carrier protein-2 ; SCPx, sterol carrier protein-x; PPARK, peroxisome proliferator activated receptor-K; RXRK, retinoid-X receptor-K. As shown recently, cytosolic and nuclear transport of free phytanic acid may be mediated by L-FABP [64], SCP2 functions in peroxisomal phytanoyl-CoA tra¤cking. The role as thiolase in pristanic acid L-oxidation is indicated.
the patient had a block in normal peroxisomal degradation of the cholesterol side chain in bile acid synthesis. A surprising ¢nding was that D-PBE de¢ciency also a¡ected peroxisomal L-oxidation of VLCFA-CoA. Although secondary causes cannot be excluded at present, this ¢nding suggests strongly that D-PBE, together with L-PBE, may play an important role also in L-oxidation of straight VLCFACoA substrates. Thus, the newly discovered D-PBE may act simultaneously on all three major substrates of peroxisomal L-oxidation (VLCFA, 2-methylbranched-chain fatty acids and bile acids) which suggests that D-PBE may in fact be the more important enzyme in human peroxisomal L-oxidation than the long-known L-PBE. More recently, positional cloning and molecular characterization of the unc-24 gene of C. elegans led to the identi¢cation of a new member of the SCP2 gene family [44]. The unc-24 gene is required for normal locomotion and interacts with genes that a¡ect the worm's response to volatile anesthetics. The predicted gene product contains a domain sim-
ilar to part of two ion channel regulators (the erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2), juxtaposed to a domain similar to SCP2. Sequence analysis suggested that the SCP2-like domain of UNC-24 is tethered to the plasma membrane by the stomatin-like domain which may be regulatory [44]. Recently, cDNA clones encoding a human homologue of UNC-24 could be isolated from a human cerebral cortex cDNA library [45]. The bipartite stomatin-like protein called hSLP-1 consists of 394 amino acids. The major stomatin-like part starts at the N-terminus whereas the SCP2-like domain is located at the C-terminal end. The SLP-1 transcript is mainly expressed in the brain, with the highest levels in the frontal lobe, cerebral cortex, caudate nucleus, amygdala, temporal lobe, putamen, substantia nigra, and hippocampus. The function of the SCP2 moiety in UNC-24 or hSLP-1 is currently unknown. 3. Role of SCP2/SCPx in peroxisomal K- and L-oxidation E¡orts in several laboratories to identify human inherited diseases that result from Scp2 mutations were not successful. However, the biological function of Scp2 was investigated by employing gene targeting in mice [17]. Besides a severe block at the level of the thiolytic cleavage in pristanic acid L-oxidation (2methyl-branched fatty acid, Fig. 2), the null mice had a block in normal peroxisomal degradation of the cholesterol side chain in bile acid synthesis [46]. A surprising ¢nding was that SCP2/SCPx de¢ciency also a¡ected peroxisomal K-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) [17]. Although secondary causes cannot be excluded at present, the data suggested strongly that the acylCoA binding function of SCP2 may play an important role in peroxisomal uptake of phytanoyl-CoA or its intraperoxisomal delivery to the phytanoyl-CoA K-hydroxylase enzyme. Conversely, no abnormalities were found for oxidation of VLCFA-CoA. The defect in normal bile acid synthesis is illustrated schematically in Fig. 3.
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Fig. 3. Role of the SCPx thiolase in bile acid synthesis. 1, 27-hydroxylation occurring in mitochondria; 2, oxidation by cytosolic alcohol dehydrogenases; 3, peroxisomal THCA-CoA oxidase; 4, 5, D-PBE; 6, SCPx. The metabolic block in SCP2/SCPx KO mice is indicated by an arrow.
4. Impact of SCP2/SCPx de¢ciency on activation of the peroxisome proliferator activated receptor, PPARK K Spontaneous peroxisome proliferation and marked alteration of gene expression in the liver are early events related to SCP2/SCPx de¢ciency in mice which may be attributed to sustained PPARK activation [47]. Similar e¡ects were reported in acyl-CoA oxidase (ACO) null mice. ACO catalyzes the ¢rst step of peroxisomal L-oxidation of VLCFA, which converts fatty acyl-CoA to 2-trans-enoyl-CoA. Mice de¢cient in ACO exhibit increased levels of VLCFA, particularly after stress with VLCFA enriched diets [48]. The block in peroxisomal L-oxidation of VLCFA is associated with steatohepatitis, increased hepatic H2 O2 levels, and hepatocellular regeneration. Similar to the SCP2 null mice, the liver of ACO null mice displayed profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by PPARK. Hepatic adenomas and carcinomas developed in ACO null mice
by 15 months of age, probably due to sustained activation of PPARK [49]. These observations implicate putative substrates for peroxisomal L-oxidation as biological ligands for PPARK. Many of the pleiotropic e¡ects that result from the ACO gene disruption resemble those which are present in SCP2 null mice, also showing spontaneous peroxisome proliferation and evidence of chronic PPARK activation. Although steatohepatitis was virtually absent in the SCP2/SCPx-de¢cient strain, hepatocarcinomas and adenomas developed shortly after the age of 1 year. It could be shown that phytanic acid serum concentrations correlate well with expression of PPARK target genes in SCP2 null mice [47]. Moreover, treatment with phytanic acid leads to pleiotropic e¡ects that could be mimicked with PPARK agonists beza¢brate and WY 14,643 but not with the retinoid-X receptor, RXRK, agonist 9-cis-retinoic acid. The ¢ndings are in line with binding of phytanic acid to a fused glutathione S-transferase-murine-PPARK ligand binding domain with almost the same a¤nity as the strong arti¢cial PPARK agonist
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WY 14,643 and phytanic acid induced expression activation of a PPRE-driven reporter gene in vitro [47]. Taken together, the currently available data provide strong support for phytanic acid acting as signal involved in direct stimulation of PPARK. This is particularly noteworthy, since direct binding or activation of rodent PPARK could so far not be demonstrated for other natural substrates of peroxisomal metabolism. In the ACO null mice, ACO de¢ciency imposes a block on VLCFA-CoA to enter the L-oxidation pathway. It is conceivable that unmetabolized VLCFA-CoA may function as biological ligands of PPARK/RXRK, leading to sustained transcriptional enhancement of genes with PPRE-containing promoters in this system. Long chain acyl-CoAs were once considered to represent a metabolic message responsible for the induction of the L-oxidation system [50,51]. This raises the question whether free fatty acids and unmetabolized synthetic peroxisome proliferators can act as direct ligands of PPARK in vivo or whether activation of this receptor is mediated by their CoA esters or downstream derivatives resulting from their L-oxidation. It is known that sulfur-substituted fatty acid derivatives and peroxisome proliferators of the ¢brate class are activated to their esters with CoA. Although these cannot enter the L-oxidation spiral, they could still function e¤ciently as peroxisome proliferators in vivo, implying that L-oxidation is not essential to generate the PPARK agonists [51,52]. On the other hand, progressive VLCFA accumulation in X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired VLCFA metabolism associated with neurological abnormalities and death during childhood, does not lead to spontaneous peroxisome proliferation in liver parenchymal cells in X-ALD patients or in mouse models for this disease, developed recently by inactivating the X-ALD gene [53^56]. Free VLCFA bind only weakly to recombinant PPARK [57,58]. In addition, dietary lipid overload, leading to increased VLCFA levels, does not induce peroxisome proliferation [53^56]. These results imply that, under in vivo conditions, the free VLCFAs are not e¡ective inducers of PPARK. The remarkable induction of spontaneous peroxisome proliferative response in ACO null mice raises the possibility that the PPARK signal-transducing event
is immediately distal to the acyl-CoA synthase-catalyzed fatty acid activation step. However, several long-chain fatty acyl-CoAs did neither bind to recombinant PPARK nor induced PPARK activation in vitro [57]. Thus, the factors that mediate peroxisome proliferation in ACO null mice are not yet clear. One possibility is that PPARK activation is mediated by a still unknown PPARK ligand that is L-oxidized within the peroxisome [59]. Such an endogenous ligand of PPARK may potentially contribute to enhanced PPARK activation in the liver of ACO null mice. Despite the fact that phytanic acid can be regarded as a bona ¢de PPARK agonist in mice, the involvement of so far unknown endogenous ligands that accumulate along with phytanic acid in the SCP2 null mice and signal PPARK activation cannot be ruled out at present. The data establish clearly that both genes, ACO and SCP2/SCPx, are required for e¤cient peroxisomal oxidation of certain fatty acids and at the same time they are key regulators of PPARK function in vivo. Thus, these mouse models may provide helpful clues in the search for so far unknown natural PPARK agonists and in screening for in vivo antagonists for this receptor. 5. Is SCP2 a cytoplasmic cholesterol carrier? Many studies have been published in which potential functions of SCP2 were investigated using assays in vitro but relatively little is known regarding the role of SCP2 in intact cells. Moncecchi et al. transfected mouse L-cell ¢broblasts with cDNAs encoding mouse pre-SCP2 and SCP2. Expression of pre-SCP2, but not of SCP2, enhanced the rate and extent of [3 H]cholesterol uptake compared to control or mock-transfected cells slightly by 1.3-fold [60]. Puglielli et al. reported that the rapid transport of de novo synthesized cholesterol to the plasma membrane was reduced after treatment with SCP2 antisense oligonucleotides of normal ¢broblasts, which suggested that the major fraction of newly synthesized cholesterol may be transported to the plasma membrane via an SCP2-dependent mechanism [15]. According to Baum et al., overexpression of SCP2 in McA-RH7777 rat hepatoma cells enhances the rate of cholesterol cycling, which reduces the availability
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of cholesterol for cholesterol ester synthesis and alters the activity of a cellular cholesterol pool involved in regulating apolipoprotein A-I-mediated high density lipoprotein cholesterol secretion. The net result of these changes was a 46% increase in plasma membrane cholesterol content [16]. In contrast, no information is currently available on the e¡ect of SCP2 overexpression on fatty acid metabolism but it appears noteworthy to us that puri¢ed SCP2 binds most fatty acids and fatty acyl-CoAs with similar or even higher a¤nity than sterols [61]. Phytanoyl-CoA binding was shown recently to be approx. 10-fold better than binding of cholesterol as measured with a highly speci¢c £uorescence resonance energy transfer assay [17,62]. Wouters et al. studied the fate of £uorescently labeled pre-SCP2 (Cy3-pre-SCP2) microinjected into BALB/c 3T3 ¢broblasts [63]. The protein colocalized to a high degree with the immuno£uorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the peroxisomal targeting signal (AKL) abolished targeting of the labeled pre-SCP2 to peroxisomes. Moreover, they investigated the association of SCP2 with peroxisomal enzymes by measuring £uorescence resonance energy transfer between the microinjected Cy3-pre-SCP2 and Cy5-labeled antibodies against the peroxisomal enzymes ACO, 3-ketoacyl-CoA thiolase, PBE, PMP70 and catalase. The data revealed a speci¢c association of SCP2 with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and PBE in the peroxisomes. These studies show a close association of SCP2 with other essential components of the peroxisomal fatty acid L-oxidation system, which supports a role of SCP2 in regulating peroxisomal L-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes. On the basis of these recent data, as well as the phenotype of the SCP2 null mice we currently favor that SCP2 functions in vivo as fatty acyl-CoA carrier in peroxisomal fatty acyl-CoA metabolism rather than in cytosolic sterol transport. Acknowledgements The authors' work was supported by grants from the Deutsche Forschungsgemeinschaft (grant Se 459/
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2), the Interdisziplina«res Zentrum fu«r Klinische Forschung, IZKF (Project A4) of the Medical Faculty, University of Mu«nster and the Bayer AG.
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Jolly, J.T. Billheimer, A.I. Scott, F. Schroeder, The sterol carrier protein-2 fatty acid binding site: an NMR, circular dichroic, and £uorescence spectroscopic determination, Biochemistry 36 (1997) 1719^1729. [62] K.W. Wirtz, F.S. Wouters, P.H. Bastiaens, R.J. Wanders, U. Seedorf, T.M. Jovin, The non-speci¢c lipid transfer protein (sterol carrier protein 2) acts as a peroxisomal fatty acylCoA binding protein, Biochem. Soc. Trans. 26 (1998) 374^ 378.
[63] F.S. Wouters, P.I. Bastiaens, K.W. Wirtz, T.M. Jovin, FRET microscopy demonstrates molecular association of non-speci¢c lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes, EMBO J. 17 (1998) 7179^7189. [64] C. Wolfrum, P. Ellinghaus, M. Fobker, U. Seedorf, G. Assmann, T. Borchers, F. Spener, Phytanic acid is ligand and transcriptional activator of murine liver fatty acid binding protein, J. Lipid Res. 40 (1999) 708^714.
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Review
Sterol carrier protein-2 Udo Seedorf *, Peter Ellinghaus, Jerzy Roch Nofer Institute for Arteriosclerosis Research, Institute for Clinical Chemistry and Laboratory Medicine, Interdisciplinary Center for Clinical Research, Westphalian Wilhelms-University, D-48149 Mu«nster, Germany Received 6 October 1999; received in revised form 9 November 1999; accepted 9 November 1999
Abstract The compartmentalization of cholesterol metabolism implies target-specific cholesterol trafficking between the endoplasmic reticulum, plasma membrane, lysosomes, mitochondria and peroxisomes. One hypothesis has been that sterol carrier protein-2 (SCP2, also known as the non-specific lipid transfer protein) acts in cholesterol transport through the cytoplasm. Recent studies employing gene targeting in mice showed, however, that mice lacking SCP2 and the related putative sterol carrier known as SCPx, develop a defect in peroxisomal L-oxidation. In addition, diminished peroxisomal K-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) in these null mice was attributed to the absence of SCP2 which has a number of properties supporting a function as carrier for fatty acyl-CoAs rather than for sterols. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Sterol carrier protein-2 ; Cholesterol; Peroxisome; L-Oxidation; Acyl-coenzyme A
1. Intracellular cholesterol tra¤cking ^ the sterol carrier hypothesis In most eukaryotic cells, the bulk of cholesterol is synthesized at the endoplasmic reticulum (ER) whereas almost 90% of the free, non-esteri¢ed fraction of this essential membrane lipid resides in the plasma membrane [1]. Cholesterol is found mainly at
Abbreviations: ACO, acyl-CoA oxidase; ACTH, adrenocorticotrophic hormone; ER, endoplasmic reticulum; LH, luteinizing hormone; NPC, Niemann-Pick disease type C; PBE, peroxisomal bifunctional enzyme; PPARK, peroxisome proliferator-activated receptor-K; SCP, sterol carrier protein; StAR, steroidogenic acute regulatory protein; VLCFA, very long chain fatty acid; X-ALD, X-linked adrenoleukodystrophy * Corresponding author. E-mail: [email protected]
the inner lea£et of the bilayer, where it limits membrane £uidity which is thought to stabilize the complex supramolecular structures that are formed between lipids, receptors, adaptor proteins and the cytoskeleton at the cell surface. The cholesterol hydroxyl group forms a hydrogen bond with a phospholipid carbonyl oxygen atom whereas the bulky steroid moiety and the £exible hydrocarbon tail are directed to the hydrophobic inner portion of the membrane. It was proposed that cholesterol is not evenly distributed within the inner lea£et of the membrane, but that it is concentrated in cholesterol-rich micro-domains called caveolae [2,3]. Caveolae are rich in sphingomyelin and VIP21 caveolin, a 21^ 24 kDa integral membrane protein that binds cholesterol at a 1:1 molar ratio [2,4^7]. The highly asymmetric distribution of cholesterol in cells makes it conceivable that intracellular tra¤cking of cholesterol requires target-speci¢c transport mechanisms that
1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 0 ) 0 0 0 4 7 - 0
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mediate its translocation from the site of synthesis at the ER to the caveolae of the plasma membrane. Moreover, one must postulate means that keep cholesterol from di¡using to the bilayer's outer lea£et and that inhibit its lateral di¡usion within the membrane. At present, not much is known about the precise mechanisms that establish the remarkable speci¢city of the machinery that regulates these fundamental processes in the cell. Speci¢c transport mechanisms have been made responsible for the transfer of cholesterol from the membranes of secondary lysosomes to the endoplasmic reticulum, the intracellular site of cholesterol esteri¢cation catalyzed by acyl-CoA cholesterol acyl transferase. A breakthrough discovery came with the identi¢cation of the genetic basis underlying Niemann-Pick disease type C (NPC), mapped to human chromosome 18 [8,9]. NPC is an inherited lipid storage disorder that a¡ects the viscera and central nervous system. Defective cholesterol tra¤cking leading to lysosomal accumulation of low density lipoprotein-derived free cholesterol is a characteristic feature of NPC cells. The mutated protein, which was named NPC1, shows homologies with the sterol sensing domains which are present in several key regulators of cholesterol homeostasis and a class of morphogen receptors called sonic hedgehog proteins (shh). NPC1 functions most likely in regulating the retroendocytic tra¤cking of cholesterol and other lysosomal cargo. Another important transport route of cholesterol, which is required mainly for the synthesis of pregnenolone and bile acids, concerns the delivery of sterols to mitochondria. Stimulation of steroid-producing cells of the gonads and adrenals with the trophic hormones luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) leads to a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the ¢rst committed step in steroid synthesis is performed by the side chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. One protein that is known to play a crucial role in this cholesterol translocation step was named steroidogenic acute regulatory protein (StAR) [10]. Lack of functional StAR causes the autosomal recessive disease
congenital lipoid adrenal hyperplasia, which is characterized by markedly impaired gonadal and adrenal steroid hormone synthesis [10]. StAR protein is expressed almost exclusively in steroid-producing cells and its presence is correlated with steroid hormone production. More recently, it could be shown that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation [11]. Little is known at present about sterol transport through the cytoplasm. Earlier studies supported that the bulk £ow of de novo synthesized cholesterol from the ER to the plasma membrane is a fast, energy-requiring process that proceeds independently of the secretory protein pathway, but may nevertheless be vesicular [12]. Alternatively, cholesterol transport through the cytoplasm could be achieved via soluble carrier proteins that would shield the lipophilic transport substrate from the aqueous phase and harbor signals that could mediate target specificity. Unfortunately, e¡orts that were aimed at isolating such proteins and cloning their corresponding cDNAs have essentially been unsuccessful. The best studied candidate for a soluble sterol carrier has been sterol carrier protein-2 (SCP2), also known as the non-speci¢c lipid transfer protein, puri¢ed almost 20 years ago on the basis of its ability to activate the enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes in vitro [13]. Because SCP2 promotes the exchange of a wide variety of sterols between membranes in vitro and its expression a¡ects sterol tra¤cking in certain tissue culture systems, it has long been thought that the protein acts as a substrate carrier in various steps of sterol metabolism [14^16]. On the other hand, low sterol transfer activity under physiologic conditions, apparent lack of speci¢city for cholesterol, and the predominant localization of SCP2 in peroxisomes made it di¤cult to understand how the protein might carry out this role in the intact cell. The purpose of this review is to summarize our current knowledge about the molecular biology of the sterol carrier protein-2 gene family and discuss potential functions of SCP2 in vivo, as studied in a murine model of complete SCP2 de¢ciency generated by gene targeting [17].
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2. The SCP2 gene family At present, the SCP2 gene family includes only four distinct members (SCP2, SCPx, D-PBE and UNC-24/hSLP-1), but it can be expected that additional homologues may be identi¢ed in the future. Apart from SCP2, which is expressed as an individual protein, the other homologues contain their SCP2 domains at the C-terminus (Fig. 1). Mammalian SCP2 is synthesized as a 143 amino acid precursor that is processed most likely in peroxisomes to the 123 amino acid mature SCP2. The human SCP2encoding gene comprises 16 exons, which span approx. 100 kb on chromosome 1p32 [18^20]. Alternate transcription initiation regulates the expression of SCP2 and a second gene product that consists of 547 amino acids (named sterol carrier protein-x, SCPx) [21]. SCPx represents a fused protein consisting of a thiolase, extending from amino acid 1 to 404, and SCP2 which is located at the carboxyl terminus [22,23]. The fused gene can be traced back to Drosophila melanogaster [24] (GenBank accession No. X97685), whereas two separated genes for SCP2 and the thiolase are present in Caenorhabditis elegans and several yeast species [25,26]. An ancient precursor of the SCP2 sequence could be identi¢ed even in the methanogenic archaeon, Methanococcus jannaschii [27]. It is known from in vitro studies that SCPx has lipid transfer activity similar to SCP2 [28]. The substrate speci¢city of the SCPx thiolase shows a preference for medium straight-chain acyl-CoA substrates, 2-methyl-branched-chain fatty acyl-CoAs (such as 3-ketopristanoyl-CoA) and bile acid precursors (such as 3K,7K,12K-trihydroxy-24-ketocholestanoyl-CoA) [29^31]. The last two substrates are not oxidized e¡ectively in mitochondria but require peroxisomal L-oxidation, which coincides with the peroxisomal localization of SCPx. The properties of the newly discovered SCPx thiolase thus di¡er clearly from the long-known peroxisomal thiolase, that was identi¢ed by Hashimoto and coworkers almost 15 years ago [32]. Unlike SCPx, this enzyme acts preferentially on the 3-keto derivatives of the straight very long chain fatty acid (VLCFA) that are metabolized in peroxisomal L-oxidation [32,33]. An interesting ¢nding was that the cDNA encoding an 80 kDa precursor cloned originally as 17L-
Fig. 1. Domain structure of the currently known members of the SCP2 gene family. Amino acid numbers of known processing sites are indicated by arrows. SCP2, sterol carrier protein-2 ; SCPx, sterol carrier protein-x; PBE, peroxisomal bifunctional enzyme; hSLP, human stomatin-like protein.
hydroxysteroid dehydrogenase type IV (17L-HSD4) contained a C-terminal domain similar to SCP2 [34]. The domain was fused to a peptide with acylCoA 2-enoyl hydratase/3-hydroxyacyl-CoA dehydrogenase activity (also known as peroxisomal bifunctional enzyme (PBE)) [35]. Processing occurs after import of the 80 kDa precursor into peroxisomes at the junction between the acyl-CoA 2-enoyl hydratase and 3-hydroxyacyl-CoA dehydrogenase domains [36]. The SCP2-like domain is required for import of the 80 kDa precursor into peroxisomes and confers a similar intrinsic lipid transfer activity to the fusion protein as was demonstrated for SCPx [37]. In contrast to the well known PBE enzyme that is also called L-bifunctional enzyme [38], because it converts trans-enoyl-CoA to their 3-keto derivatives via the L-hydroxy stereoisomer, the new PBE enzyme catalyzes the same transformation via the D-stereoisomer [39,40]. Therefore, Hashimoto and coworkers have introduced the name `D-bifunctional enzyme' for the peroxisomal protein that was originally cloned as 17L-HSD4. The two PBE proteins di¡er in their substrate speci¢city. Whereas the D-PBE catalyzes the formation of 3-ketoacyl-CoA intermediates from straight and 2-methyl-branched-chain fatty acyl-CoAs, the activity of L-PBE is only high with the straight-chain substrates [39^42]. Convincing evidence showing that these ¢ndings are relevant also in vivo were obtained by van Grunsven et al. who identi¢ed a patient with isolated D-PBE de¢ciency [43]. Besides a severe block in pristanic acid L-oxidation,
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Fig. 2. Schematic illustration of cellular phytanic acid tra¤cking and metabolism. L-FABP, liver fatty acid binding protein ; SCP2, sterol carrier protein-2 ; SCPx, sterol carrier protein-x; PPARK, peroxisome proliferator activated receptor-K; RXRK, retinoid-X receptor-K. As shown recently, cytosolic and nuclear transport of free phytanic acid may be mediated by L-FABP [64], SCP2 functions in peroxisomal phytanoyl-CoA tra¤cking. The role as thiolase in pristanic acid L-oxidation is indicated.
the patient had a block in normal peroxisomal degradation of the cholesterol side chain in bile acid synthesis. A surprising ¢nding was that D-PBE de¢ciency also a¡ected peroxisomal L-oxidation of VLCFA-CoA. Although secondary causes cannot be excluded at present, this ¢nding suggests strongly that D-PBE, together with L-PBE, may play an important role also in L-oxidation of straight VLCFACoA substrates. Thus, the newly discovered D-PBE may act simultaneously on all three major substrates of peroxisomal L-oxidation (VLCFA, 2-methylbranched-chain fatty acids and bile acids) which suggests that D-PBE may in fact be the more important enzyme in human peroxisomal L-oxidation than the long-known L-PBE. More recently, positional cloning and molecular characterization of the unc-24 gene of C. elegans led to the identi¢cation of a new member of the SCP2 gene family [44]. The unc-24 gene is required for normal locomotion and interacts with genes that a¡ect the worm's response to volatile anesthetics. The predicted gene product contains a domain sim-
ilar to part of two ion channel regulators (the erythrocyte integral membrane protein stomatin and the C. elegans neuronal protein MEC-2), juxtaposed to a domain similar to SCP2. Sequence analysis suggested that the SCP2-like domain of UNC-24 is tethered to the plasma membrane by the stomatin-like domain which may be regulatory [44]. Recently, cDNA clones encoding a human homologue of UNC-24 could be isolated from a human cerebral cortex cDNA library [45]. The bipartite stomatin-like protein called hSLP-1 consists of 394 amino acids. The major stomatin-like part starts at the N-terminus whereas the SCP2-like domain is located at the C-terminal end. The SLP-1 transcript is mainly expressed in the brain, with the highest levels in the frontal lobe, cerebral cortex, caudate nucleus, amygdala, temporal lobe, putamen, substantia nigra, and hippocampus. The function of the SCP2 moiety in UNC-24 or hSLP-1 is currently unknown. 3. Role of SCP2/SCPx in peroxisomal K- and L-oxidation E¡orts in several laboratories to identify human inherited diseases that result from Scp2 mutations were not successful. However, the biological function of Scp2 was investigated by employing gene targeting in mice [17]. Besides a severe block at the level of the thiolytic cleavage in pristanic acid L-oxidation (2methyl-branched fatty acid, Fig. 2), the null mice had a block in normal peroxisomal degradation of the cholesterol side chain in bile acid synthesis [46]. A surprising ¢nding was that SCP2/SCPx de¢ciency also a¡ected peroxisomal K-oxidation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) [17]. Although secondary causes cannot be excluded at present, the data suggested strongly that the acylCoA binding function of SCP2 may play an important role in peroxisomal uptake of phytanoyl-CoA or its intraperoxisomal delivery to the phytanoyl-CoA K-hydroxylase enzyme. Conversely, no abnormalities were found for oxidation of VLCFA-CoA. The defect in normal bile acid synthesis is illustrated schematically in Fig. 3.
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Fig. 3. Role of the SCPx thiolase in bile acid synthesis. 1, 27-hydroxylation occurring in mitochondria; 2, oxidation by cytosolic alcohol dehydrogenases; 3, peroxisomal THCA-CoA oxidase; 4, 5, D-PBE; 6, SCPx. The metabolic block in SCP2/SCPx KO mice is indicated by an arrow.
4. Impact of SCP2/SCPx de¢ciency on activation of the peroxisome proliferator activated receptor, PPARK K Spontaneous peroxisome proliferation and marked alteration of gene expression in the liver are early events related to SCP2/SCPx de¢ciency in mice which may be attributed to sustained PPARK activation [47]. Similar e¡ects were reported in acyl-CoA oxidase (ACO) null mice. ACO catalyzes the ¢rst step of peroxisomal L-oxidation of VLCFA, which converts fatty acyl-CoA to 2-trans-enoyl-CoA. Mice de¢cient in ACO exhibit increased levels of VLCFA, particularly after stress with VLCFA enriched diets [48]. The block in peroxisomal L-oxidation of VLCFA is associated with steatohepatitis, increased hepatic H2 O2 levels, and hepatocellular regeneration. Similar to the SCP2 null mice, the liver of ACO null mice displayed profound generalized spontaneous peroxisome proliferation and increased mRNA levels of genes that are regulated by PPARK. Hepatic adenomas and carcinomas developed in ACO null mice
by 15 months of age, probably due to sustained activation of PPARK [49]. These observations implicate putative substrates for peroxisomal L-oxidation as biological ligands for PPARK. Many of the pleiotropic e¡ects that result from the ACO gene disruption resemble those which are present in SCP2 null mice, also showing spontaneous peroxisome proliferation and evidence of chronic PPARK activation. Although steatohepatitis was virtually absent in the SCP2/SCPx-de¢cient strain, hepatocarcinomas and adenomas developed shortly after the age of 1 year. It could be shown that phytanic acid serum concentrations correlate well with expression of PPARK target genes in SCP2 null mice [47]. Moreover, treatment with phytanic acid leads to pleiotropic e¡ects that could be mimicked with PPARK agonists beza¢brate and WY 14,643 but not with the retinoid-X receptor, RXRK, agonist 9-cis-retinoic acid. The ¢ndings are in line with binding of phytanic acid to a fused glutathione S-transferase-murine-PPARK ligand binding domain with almost the same a¤nity as the strong arti¢cial PPARK agonist
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WY 14,643 and phytanic acid induced expression activation of a PPRE-driven reporter gene in vitro [47]. Taken together, the currently available data provide strong support for phytanic acid acting as signal involved in direct stimulation of PPARK. This is particularly noteworthy, since direct binding or activation of rodent PPARK could so far not be demonstrated for other natural substrates of peroxisomal metabolism. In the ACO null mice, ACO de¢ciency imposes a block on VLCFA-CoA to enter the L-oxidation pathway. It is conceivable that unmetabolized VLCFA-CoA may function as biological ligands of PPARK/RXRK, leading to sustained transcriptional enhancement of genes with PPRE-containing promoters in this system. Long chain acyl-CoAs were once considered to represent a metabolic message responsible for the induction of the L-oxidation system [50,51]. This raises the question whether free fatty acids and unmetabolized synthetic peroxisome proliferators can act as direct ligands of PPARK in vivo or whether activation of this receptor is mediated by their CoA esters or downstream derivatives resulting from their L-oxidation. It is known that sulfur-substituted fatty acid derivatives and peroxisome proliferators of the ¢brate class are activated to their esters with CoA. Although these cannot enter the L-oxidation spiral, they could still function e¤ciently as peroxisome proliferators in vivo, implying that L-oxidation is not essential to generate the PPARK agonists [51,52]. On the other hand, progressive VLCFA accumulation in X-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder with impaired VLCFA metabolism associated with neurological abnormalities and death during childhood, does not lead to spontaneous peroxisome proliferation in liver parenchymal cells in X-ALD patients or in mouse models for this disease, developed recently by inactivating the X-ALD gene [53^56]. Free VLCFA bind only weakly to recombinant PPARK [57,58]. In addition, dietary lipid overload, leading to increased VLCFA levels, does not induce peroxisome proliferation [53^56]. These results imply that, under in vivo conditions, the free VLCFAs are not e¡ective inducers of PPARK. The remarkable induction of spontaneous peroxisome proliferative response in ACO null mice raises the possibility that the PPARK signal-transducing event
is immediately distal to the acyl-CoA synthase-catalyzed fatty acid activation step. However, several long-chain fatty acyl-CoAs did neither bind to recombinant PPARK nor induced PPARK activation in vitro [57]. Thus, the factors that mediate peroxisome proliferation in ACO null mice are not yet clear. One possibility is that PPARK activation is mediated by a still unknown PPARK ligand that is L-oxidized within the peroxisome [59]. Such an endogenous ligand of PPARK may potentially contribute to enhanced PPARK activation in the liver of ACO null mice. Despite the fact that phytanic acid can be regarded as a bona ¢de PPARK agonist in mice, the involvement of so far unknown endogenous ligands that accumulate along with phytanic acid in the SCP2 null mice and signal PPARK activation cannot be ruled out at present. The data establish clearly that both genes, ACO and SCP2/SCPx, are required for e¤cient peroxisomal oxidation of certain fatty acids and at the same time they are key regulators of PPARK function in vivo. Thus, these mouse models may provide helpful clues in the search for so far unknown natural PPARK agonists and in screening for in vivo antagonists for this receptor. 5. Is SCP2 a cytoplasmic cholesterol carrier? Many studies have been published in which potential functions of SCP2 were investigated using assays in vitro but relatively little is known regarding the role of SCP2 in intact cells. Moncecchi et al. transfected mouse L-cell ¢broblasts with cDNAs encoding mouse pre-SCP2 and SCP2. Expression of pre-SCP2, but not of SCP2, enhanced the rate and extent of [3 H]cholesterol uptake compared to control or mock-transfected cells slightly by 1.3-fold [60]. Puglielli et al. reported that the rapid transport of de novo synthesized cholesterol to the plasma membrane was reduced after treatment with SCP2 antisense oligonucleotides of normal ¢broblasts, which suggested that the major fraction of newly synthesized cholesterol may be transported to the plasma membrane via an SCP2-dependent mechanism [15]. According to Baum et al., overexpression of SCP2 in McA-RH7777 rat hepatoma cells enhances the rate of cholesterol cycling, which reduces the availability
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of cholesterol for cholesterol ester synthesis and alters the activity of a cellular cholesterol pool involved in regulating apolipoprotein A-I-mediated high density lipoprotein cholesterol secretion. The net result of these changes was a 46% increase in plasma membrane cholesterol content [16]. In contrast, no information is currently available on the e¡ect of SCP2 overexpression on fatty acid metabolism but it appears noteworthy to us that puri¢ed SCP2 binds most fatty acids and fatty acyl-CoAs with similar or even higher a¤nity than sterols [61]. Phytanoyl-CoA binding was shown recently to be approx. 10-fold better than binding of cholesterol as measured with a highly speci¢c £uorescence resonance energy transfer assay [17,62]. Wouters et al. studied the fate of £uorescently labeled pre-SCP2 (Cy3-pre-SCP2) microinjected into BALB/c 3T3 ¢broblasts [63]. The protein colocalized to a high degree with the immuno£uorescence pattern for the peroxisomal enzyme acyl-CoA oxidase. Proteolytic removal of the C-terminal leucine of the peroxisomal targeting signal (AKL) abolished targeting of the labeled pre-SCP2 to peroxisomes. Moreover, they investigated the association of SCP2 with peroxisomal enzymes by measuring £uorescence resonance energy transfer between the microinjected Cy3-pre-SCP2 and Cy5-labeled antibodies against the peroxisomal enzymes ACO, 3-ketoacyl-CoA thiolase, PBE, PMP70 and catalase. The data revealed a speci¢c association of SCP2 with acyl-CoA oxidase, 3-ketoacyl-CoA thiolase and PBE in the peroxisomes. These studies show a close association of SCP2 with other essential components of the peroxisomal fatty acid L-oxidation system, which supports a role of SCP2 in regulating peroxisomal L-oxidation, e.g. by facilitating the presentation of substrates and/or stabilization of the enzymes. On the basis of these recent data, as well as the phenotype of the SCP2 null mice we currently favor that SCP2 functions in vivo as fatty acyl-CoA carrier in peroxisomal fatty acyl-CoA metabolism rather than in cytosolic sterol transport. Acknowledgements The authors' work was supported by grants from the Deutsche Forschungsgemeinschaft (grant Se 459/
51
2), the Interdisziplina«res Zentrum fu«r Klinische Forschung, IZKF (Project A4) of the Medical Faculty, University of Mu«nster and the Bayer AG.
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