- Email: [email protected]
The Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function
36 Chandrashekar, V. and Bartke, A. (1993) Effects of age and endogenously secreted human GH on the regulation of gonadotropin secretion in female and male transgenic mice expressing the human growth hormone gene. Endocrinology 132, 1482–1488 37 Chandrashekar, V. and Bartke, A. (1998) The role of growth hormone in the control of gonadotropin secretion in adult male rats. Endocrinology 139, 1067–1074 38 Childs, G.V. et al. (1994) Cells that express luteinizing hormone (LH) and follicle stimulating hormone (FSH) beta (b) subunit mRNAs during the estrous cycle: the major contributors contain LHb, FSHb and/or
growth hormone. Endocrinology, 134, 990–997 39 Childs, G.V. et al. (1994) Cytochemical detection of GnRH binding sites on rat pituitary cells with LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134, 1943–1951 40 Childs, G.V. et al. (1997) Differential effects of inhibin on gonadotropin stores and gonadotropin releasing hormone binding to pituitary cells from cycling female rats. Endocrinology 138, 1577–1584 41 Childs, G.V. and Unabia, G. (1997) Cytochemical studies of the effects of activin on gonadotropin releasing hormone (GnRH) binding by pituitary gonadotropes and
The Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function Ann Vogel Hertzel and David A. Bernlohr
Intracellular fatty acid-binding proteins associate with fatty acids and other hydrophobic biomolecules in an internal cavity, providing for solubilization and metabolic trafficking. Analyses of their in vivo function by molecular and genetic techniques reveal specific function(s) that fatty acid-binding proteins perform with respect to fatty acid uptake, oxidation and overall metabolic homeostasis. Intracellular fatty acid-binding proteins (FABPs) are members of a multigene family encoding ~15-kDa proteins, which bind a hydrophobic ligand in a non-covalent, reversible manner (reviewed in Refs 1–3). The nine family members have between 20% and 70% identity in their amino acid sequence. Despite the wide variance in primary sequence, numerous X-ray crystal structures have shown a common tertiary fold forming a b-barrel (Fig. 1). The barrel comprises ten antiparallel bstrands, linked by hydrogen bonds, which are organized into two nearly orthogonal b-sheets. Importantly, the A. Vogel Hertzel and D.A. Bernlohr are at the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St Paul, MN 55108, USA. Tel: 11 612 624 2712, Fax: 11 612 625 5780, email: [email protected]. umn.edu
TEM Vol. 11, No. 5, 2000
b-sheets create an internal, water-filled cavity, lined with ~50% polar amino acids. Although the cavity is significantly larger (two-three times) than the
growth hormone cells. J. Histochem. Cytochem. 45, 1603–1610 42 Childs, G.V. et al. (1999) Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats. J. Endocrinol. 162, 177–187 43 Childs, G.V. et al. (2000) Differential expression of growth hormone mRNA by somatotropes and gonadotropes in male and cycling female rats. Endocrinology 141, 1560–1570 44 Lee, B.L. et al. (1993) Expression of follistatin mRNA in somatotropes and mammotropes early in the estrous cycle. J. Histochem. Cytochem. 41, 955–960
volume of a fatty acid, typically only a single fatty acid is bound in the cavity, with the carboxylate group oriented inwards, coordinated by a tyrosine and two arginine residues. At the N-terminus of FABPs, a helix–loop–helix motif forms a cap-like structure on the ‘top’ of the barrel. Because there is no obvious opening for the fatty acids to enter or exit the cavity, the portal hypothesis was proposed. This involves a transient conformational change around the helix–loop–helix area and adjacent loops connecting b-strands, thereby allowing the fatty acid to enter or exit the cavity4 (Fig. 1). The function(s) of these proteins within cells has remained elusive. Although the in vitro binding of a fatty acid has been analyzed extensively, the in vivo function is less well defined. It has been hypothesized that owing to the low solubility of fatty acids in an
Figure 1. Ribbon diagram of the X-ray crystal structure of fatty acid-binding protein 4 (FABP4). (a) Oleate ligand (yellow) bound in the cavity of FABP4 with the helix–loop–helix motif in top part of the figure. b-Strands A–E are in front and b-strands F–J are behind. (b) ‘Top’ view of FABP4, looking down on the portal region. The portal is surrounded by the second a-helix, and loops (noted with arrows) between b-strands C and D, and b-strands E and F. This figure was created with the use of RasMol v2.6 with data from the Brookhaven Protein DataBank, Id# 1LID.
1043-2760/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1043-2760(00)00257-5
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aqueous environment the FABPs are required to facilitate their solubilization. Additional proposed functions include facilitating the influx of fatty acids across the plasma membrane (PM) (preventing efflux by intracellular association), transport of the fatty acids within the cell, determining compartmentalization for storage, modulation of activity of enzymes involved in fatty acid metabolism and protection of enzymes and membranes from detergent-like effects of fatty acids. The question that arises then is: why are there nine different members seemingly performing the same function? A potential clue lies in the unique tissue-specific expression patterns of each member of the family (Table 1), implying that the proteins might be carrying out specialized functions, thereby justifying the need for a variety of family members. The use of molecular and genetic methods to analyze FABP functions has, in some cases, shed light upon the functional aspects of some FABPs. Because many of these proteins have been cloned from various organisms, and from different cell or tissue types, each has been named according to the location studied. Owing to the many names for the same or orthologous proteins, we will refer to each FABP only by its gene name (Table 1). • FABP1 FABP1 is found in liver, intestine and kidney and is unique in that it can bind two ligands, the first in the more typical configuration of carboxyl group inward and the second in the opposite orientation, with the carboxyl group outward5. In addition to binding long-chain fatty acids, it binds fatty acyl-CoAs, peroxisome proliferators, prostaglandins, bile acids, bilirubin, hydroxy and hydroperoxy metabolites of fatty acids, lysophosphatidic acids, selenium, heme and other hydrophobic ligands6. Transfection of Fabp1 into fibroblasts stimulated the initial rate and extent of fatty acid uptake and increased esterification into specific lipid pools7,8. In HepG2 cells, peroxisome proliferators increased FABP1 expression as well as stimulating fatty acid uptake. Stable transfection of antisense
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Table 1. The fatty acid-binding protein multigene familya Gene name
Common name
Other names (species)
FABP1
Liver
FABP2
Intestinal
FABP3
Heart/muscle
FABP4
Adipocyte
FABP5
Epidermal
FABP6
Ileal
FABP7
Brain
FABP9
Testis
MP2
Myelin P2
L-FABP h-FABP Z protein I-FABP gFABP H-FABP M-FABP MDGI c-FABP ALBP aP2 A-FABP p422 p15 Klbp (murine) Mal-1 (murine) E-FABP (human) PA-FABP (human) C-Fabp (rat) S-Fabp (rat) Le-Lbp (rat) Da11 (rat) LP2 (bovine) Melanogenic inhibitor IlLBP ILBP BABP IL-FABP I15P Gastrotropin B-FABP BLBP MRG R-FABP (chicken) T-FABP TLBP PERF15 Myelin P2 PMP2 MLBP My-FABP
Expression Liver, intestine, kidney, lung(est)
Intestine Heart, mammary, skeletal muscle
Adipose tissue, macrophages
Abnormal skin, lens, adipose tissue, endothelial cells, lung, mammary cells, brain, stomach, tongue, stomach, placenta, heart, skeletal muscle, intestine, testis, retina
Ileum
Brain, olfactory bulb
Testis
Schwann cells
a Primary references for this table and explanations for all names given above can be found at the fatty acidbinding protein web site: http://cbs.umn.edu/bmbb/FABP.html
RNA reduced FABP1 expression by 84% and reduced the rate of oleate uptake by 66% (Ref. 9). Consistent with a role for FABP1 in uptake, growth-hormone injections of rats increased FABP1 levels and fatty acid uptake, whereas hypophysectomized rats had a decrease in FABP1 levels, which also correlated with a decrease in fatty acid uptake10–12.
• FABP2 FABP2 has been studied intensively since the initial reporting that the A54T polymorphism in FABP2 alleles of Pima Indians was linked to a predisposition to type 2 diabetes13. This single amino acid substitution doubles the affinity of FABP2 for fatty acids and increases fatty acid uptake14. There is a general TEM Vol. 11, No. 5, 2000
correlation of FABP2 A54T with bodymass index (BMI), fasting triglycerides, high basal insulin levels, but not diabetes mellitus in obese Japanese15–17, aboriginal Canadians18 and Guadeloupe Indian populations19. In subsequent studies of Pima Indians, A54T was not associated with insulin resistance, but with a high lipid oxidation rate and raised high-density lipoprotein (HDL) and low-density lipoprotein (LDL) triglycerides20. Several systems were used to correlate FABP2 levels with fatty acid uptake. Fabp2 transfected into mouse fibroblasts or differentiated intestinal cells did not increase fatty acid uptake, but increased esterification into specific lipid pools7,21,22. Undifferentiated mouse pluripotent embryonic stem cells transfected with Fabp2 increased fatty acid uptake 1.7-fold and increased the intracellular diffusion constant similarly. Differentiation of these embryonic stem cells into fibroblast-like cells resulted in reduced FABP2 levels and decreased fatty acid uptake23. Furthermore, in a human enterocyte cell line, Caco-2, treatment with epidermal growth factor (EGF) decreases levels of FABP2 and inhibits fatty acid uptake24. Therefore, in all of these cases, the levels of FABP2 correlated with the ability of various cell lines to take up fatty acids. • FABP3 The heart typically derives 70% of its energy requirements from the oxidation of fat, even more upon fasting or when in a diabetic state. Owing to the high demand for lipids in the heart, FABP3 has been proposed to play a role in transporting fatty acids from the sarcolemma to their intracellular sites of metabolism. Not only has FABP3 been shown to be expressed abundantly in cardiomyocytes, and at lower levels in other heart cells, but it is also expressed in red skeletal muscles, renal cortex, testis and brain25. FABP3 levels are influenced by testosterone (increased by 33%), endurance training (increased by 29%) and the circadian rhythm (increased by 100% in dark over light). Changes are also seen in pathophysiological conditions; those that tend to increase fatty acid oxidation increase TEM Vol. 11, No. 5, 2000
FABP3 (Ref. 25). In an attempt to define the role of FABP3 in lipid metabolism more fully, transfection of cell lines that do not contain FABP3 has yielded mixed results. In 3T3 pre-adipocytes and COS7 cells, transfection of Fabp3 cDNA did not alter fatty acid uptake26. However, in a human breast cancer line, MCF7, transient expression of bovine Fabp3 increased uptake of oleate 67%. The increase in uptake might be underestimated owing to the presence of FABP5 in MCF7 cells27. To test the function of FABP3 in vivo, a disruption of the Fabp3 locus in 129 BALB/c mice was created28. These mice are fertile and have no obvious phenotypic abnormalities. Histological sections of heart, skeletal muscle and liver tissues from three-month old mice appeared normal. Fatty acid influx experiments showed the first differences: cardiac uptake of long-chain fatty acids in Fabp3 null mice was dramatically decreased compared with wild-type mice. The specificity in the defect of fatty acid uptake was tested by comparing the initial rate of uptake for a fatty acid bound by FABP3 (palmitate) to that of a non-ligand (octanoate). The rate of palmitate uptake was reduced by 45% in cardiac myocytes of FABP3-deficient mice; in contrast, uptake of octanoate was unaltered. Owing to the inability to internalize and perhaps transport fatty acids efficiently, the heart muscle switches to glucose utilization as an energy source, as confirmed by the dramatic increase in glucose transport (Fig. 2)28. Further analysis of the heart demonstrated no change in the levels of putative fatty acid transport proteins, acyl-CoA synthetase, or acyl-CoA-binding protein. The molecular defect appears to be before the b-oxidation step because the fatty acid oxidative capacities of heart homogenates were unaltered if the fatty acids were delivered on albumin29. In resting mice, this change in energy source appeared to be tolerated. However, when FABP3 null mice were stressed with the higher energy demands of exercise, these mice exhibited exercise intolerance, as indicated by higher levels of exhaustion and even
death. As such, FABP3 appears to be required for efficient cardiac fatty acid transport and oxidation28. • FABP4 To investigate the in vivo function of FABP4, a targeted disruption of the locus was made in C57BL/6J mice30. These mice appeared healthy and seemed to develop adipocytes normally. The lack of a phenotypic change prompted a search for the expression of genes encoding other FABPs in adipocytes. Another member of the FABP gene family, Fabp5, was upregulated in adipocytes 20–40-fold (mRNA), with the protein being increased 13fold30,31. When the mice were stressed with diet-induced obesity, fasting plasma glucose, insulin and cholesterol levels were decreased relative to wildtype mice, even though the mice gained a similar amount of weight. The serum triglycerides in obese wild-type mice were significantly raised relative to obese FABP4 null mice, whose serum triglycerides remained low, potentially because of an alteration in triglyceride synthesis and/or secretion. Glucose and insulin tolerance tests indicated the maintenance of insulin sensitivity in obese FABP4 null mice, contrasting
FABP3 +/+ G
FABP3 –/–
FA FA
G
G
FA PM
G
FA FABP3
β-Oxidation Fuel: FA
G G
G G
G G
G
Glycolysis Fuel: G
trends in Endocrinology and Metabolism
Figure 2. Schematic comparison of cardiac cells from wild-type (Fabp31/1) and Fabp32/2 null mice, demonstrating the change in energy source. Because they cannot properly influx and oxidize FAs, FABP3 null mice switch to glucose utilization replacing the normal use of fatty acids. Abbreviations: FA, fatty acid; FABP3, fatty acid-binding protein 3; G, glucose molecule; PM, plasma membrane.
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FA FA PM Uptake
Esterification? Metabolism?
FABP4
Lipolysis
FA FABP4
Insulin resistance?
HSL Triglyceride droplet FA
trends in Endocrinology and Metabolism
Figure 3. Schematic model of intracellular transport of FAs by FABP4. Functions include binding of FAs, interaction with HSL, the delivery of FAs for esterification and/or other metabolism, release of FAs from lipolysis and providing a link to insulin resistance. Abbreviations: FA, fatty acid; FABP4, fatty acid-binding protein 4; HSL, hormone-sensitive lipase; PM, plasma membrane.
with the insulin resistance that occurred in the wild-type mice. Thus, a single disruption in FABP4 created a genetic model to unlink obesity and insulin resistance. Changes in lipid metabolism were manifested as a threefold increase in the total amount of free fatty acids in adipocytes. Consistent with this, a decrease of ~40% in basal and stimulated lipolysis was also noted, without a change in the production of hormone-sensitive lipase or perilipin. The composition of the fatty acids in the adipocyte remained unaltered, whereas there were a few small but significant differences in the fatty acid composition of the sera30,31. Another indication of the function of FABP4 came from the finding that FABP4 interacts directly with hormonesensitive lipase32 (Fig. 3). Originally identified through yeast two-hybrid assays, the interaction was confirmed with other experiments testing protein– protein interactions, including glutathione S-transferase (GST)-pulldowns and co-immunoprecipitations of the hormone-sensitive lipase (HSL)–FABP4
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complex. The interaction domain of HSL resides in the N-terminal portion of the protein, whereas the catalytic domain has been localized to the C-terminus. Although several interpretations are possible, this interaction is consistent with a role for FABP4 in facilitating fatty acid trafficking in response to lipolytic stimulation. These results suggest FABP4 functions to traffic fatty acids away from the triglyceride after hydrolysis. It is still unclear exactly where FABP4 is taking the fatty acid: to the PM for efflux or to internal membranes for re-esterification? • FABP5 Several types of cells or tissues have been reported to express the epidermal Fabp5 (Table 1), including lens, adipose, mammary and endothelial cells, stratified epithelia of epidermis and tongue, stomach, heart, brain, liver, spleen, muscle, lung, intestine, bone marrow, renal medulla, testis, urothelium and retina33,34. Binding studies have been used to elucidate ligands for FABP5; these include long-chain fatty acids but do not include retinoic acid2,34,35. FABP5 has been shown to be upregulated in a number of altered states with disturbed lipid profiles. It is highly upregulated in benign papillomas, carcinomas36, skin warts and psoriatic keratinocytes (an increase of approximately 80-fold)35,37. In addition, it is upregulated in bladder transitional-cell carcinomas38 and peripheral nerve trauma39. Increased Fabp5 expression is seen in the hippocampal glia of the brain after systemic treatment with the seizurecausing chemical kainic acid40. In addition, sodium dodecyl sulfate (SDS) or acetone treatment of human skin, known to disturb the barrier function as seen by transepidermal water loss, upregulates FABP5 production41–43. Independent of development or differentiation, this FABP exhibits the most highly regulated synthesis of all FABP forms. However, the specific function(s) for FABP5 in the various cell types remains undefined. • FABP6 FABP6 is found in the ileum, ovary and adrenal gland44,45, and although it is homologous to the other members of the
family, it has low or no binding affinity for long-chain fatty acids, but has high affinity for bile acids. It has been hypothesized to function as a cytosolic receptor for bile acids transported by the sodiumdependent action of the ileal bile-acid transporter46. Recently, bile acids have been shown to regulate the expression of Fabp6 (Ref. 47) and this regulation is achieved through the actions of the farnesoid-X-receptor (FXR), an orphan receptor whose physiological ligands are bile acids48. • FABP7 Fabp7 is expressed at the highest levels in the developing brain and retina, with decreased levels in the adult49. FABP7 binds very-long-chain fatty acids, such as docosahexaenoic acid with very high affinity, ~20-fold greater than any longchain fatty acid50. Of these, it will bind the longer C18–C20, but not C16 fatty acids, and seems to prefer unsaturated to saturated fatty acids. In addition, it has been shown to bind oleoyl-CoA as well as lysophosphotidic acid51. The most probable in vivo ligand is docosahexaenoic acid, because it is bound with high affinity and the requirement for docosahexaenoic acid for proper brain development corresponds to the same time period in which Fabp7 is expressed50. FABP7 might specifically solubilize the very-long-chain fatty acids, thereby allowing for proper uptake and intracellular trafficking. • Myelin P2 P2 is expressed in Schwann cells and is one of the major proteins of peripheral myelin. P2 avidly associates with membrane phospholipids, and because 70% of myelin is composed of lipids, it is postulated that P2 is a lipid carrier involved in the production and maintenance of myelin. In vitro binding studies indicated high affinity for oleic acid, retinoic acid and retinol52. Purification of P2 from myelin identified the relative profile of myelin lipids bound by P2 to be similar to that present in myelin, with some alterations in the relative ratios53. • Conclusion In 1987, Sweetser et al.54 wrote that FABPs were ‘abundant proteins in search TEM Vol. 11, No. 5, 2000
of a function’. The advent of the knockout mouse and other molecular technologies has strongly implicated FABPs as more than simple cellular fatty acid buffers; they are now thought to be active fatty acid chaperones. The proteins not only protect and shuttle fatty acids within the cell, but actively participate in the acquisition or removal of fatty acids from intracellular sites, contributing specificity through selection in ligand binding. Because transgenic and knockout studies have provided the most informative results indicating potential role(s) of FABPs in lipid metabolism, the development of additional null lines will probably provide further insights into the functions of these ubiquitous fatty acid chaperones. • Acknowledgements We thank members of the Bernlohr laboratory for helpful suggestions and comments during the preparation of this manuscript. This work was supported by grants NSF 9816575, NIH DK 53189 and NRSA 1 F32 DK09599-01. References 1 Glatz, J.F. and van der Vusse, G.J. (1996) Cellular fatty acid-binding proteins: their function and physiological significance. Prog. Lipid Res. 35, 243–282 2 Bernlohr, D.A. et al. (1997) Intracellular lipid-binding proteins and their genes. Annu. Rev. Nutr. 17, 277–303 3 Coe, N.R. and Bernlohr, D.A. (1998) Physiological properties and functions of intracellular fatty acid-binding proteins. Biochim. Biophys. Acta 1391, 287–306 4 Sacchettini, J.C. et al. (1989) Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. J. Mol. Biol. 208, 327–339 5 Thompson, J. et al. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates. J. Biol. Chem. 272, 7140–7150 6 Keler, T. et al. (1997) Liver fatty acid binding protein and mitogenesis in transfected hepatoma cells. Adv. Exp. Med. Biol. 400A, 517–524 7 Prows, D.R. et al. (1995) Intestinal and liver fatty acid-binding proteins differentially affect fatty acid uptake and esterification in L-cells. Lipids 30, 907–910 8 Murphy, E.J. et al. (1996) Liver fatty acidbinding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism. Biochim. Biophys. Acta 1301, 191–198 9 Wolfrum, C. et al. (1999) Variation of livertype fatty acid-binding protein content in the human hepatoma cell line HepG2 by TEM Vol. 11, No. 5, 2000
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Sterol 27-hydroxylase Deficiency: A Rare Cause of Xanthomas in Normocholesterolemic Humans Ingemar Björkhem and Eran Leitersdorf
Cerebrotendinous xanthomatosis is characterized by the accumulation of cholestanol and cholesterol in xanthomas and brain causing a number of severe symptoms. More than 20 different mutations have been identified in the gene encoding sterol 27-hydroxylase. Defects in the gene lead to reduced bile acid biosynthesis, with accumulation of 7a-hydroxylated intermediates, one of which is a precursor to cholestanol. The disease can be treated successfully with chenodeoxycholic acid, which reduces the upregulation of cholesterol 7a-hydroxylase and, therefore, the formation of cholestanol. Disruption of the gene encoding sterol 27-hydroxylase in mice does not have the same metabolic consequences as in humans.
Familial hypercholesterolemia is by far the most common cause of xanthomas. However, among patients with xanthomas, there are two small subgroups in which increased levels of cholesterol in the circulation are not the primary I. Björkhem is at the Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden; and E. Leitersdorf is at the Department of Medicine, Hadassah University Hospital, IL-91120 Jerusalem, Israel. Tel: 146 8 585 812 35, Fax: 146 8 585 812 60, email: [email protected]
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cause for xanthomas development. These two rare sterol storage diseases are cerebrotendinous xanthomatosis (CTX) and phytosterolemia. CTX has now been characterized at the gene level and is reviewed here. For a more extensive review, see Ref. 1. • Phenotype CTX was first described by Van Bogaert et al. in 1937 (Ref. 2). The patient they described suffered from dementia, ataxia, cataracts and xanthomas in the tendons and nervous system. Several hundred
50 Xu, L.Z. et al. (1996) Ligand specificity of brain lipid-binding protein. J. Biol. Chem. 271, 24711–24719 51 Myers-Payne, S.C. et al. (1996) Isolation and characterization of two fatty acid binding proteins from mouse brain. J. Neurochem. 66, 1648–1656 52 Uyemura, K. et al. (1984) Lipid binding activities of the P2 protein in peripheral nerve myelin. Neurochem. Res. 9, 1509–1514 53 Riccio, P. et al. (1998) Purification of bovine P2 myelin protein with bound lipids. NeuroReport 9, 2769–2773 54 Sweetser, D.A. et al. (1987) The metabolic significance of mammalian fatty-acidbinding proteins: abundant proteins in search of a function. Annu. Rev. Nutr. 7, 337–359
patients have since been described, with most cases being in Japan, Israel and The Netherlands (so far, no cases have been diagnosed in Australia). In CTX patients, cholesterol levels were raised in the tissues but not in the blood, and the xanthomas were shown to contain high levels of cholestanol, the 5a-saturated analog of cholesterol3. The major clinical manifestations of CTX are caused by the accumulation of cholestanol and cholesterol in almost every tissue, including the central nervous system (CNS). • Characterization of CTX and the Role of the Defective Enzyme In 1974, Setoguchi et al. made the key discovery that CTX patients have a defect in bile-acid biosynthesis, with incomplete oxidation of the C27-steroid side chain4. In 1980, Oftebro et al. reported that a liver biopsy from a patient with CTX had an almost complete lack of mitochondrial sterol 27-hydroxylase5. Sterol 27-hydroxylase is a mitochondrial cytochrome P-450 species (CYP27) that is essential for the normal degradation of the steroid side chain in connection with formation of bile acids from cholesterol. Sterol 27-hydroxylase catalyzes the first step in the normal oxidation of the steroid side chain, converting cholesterol, as well as different 7a-hydroxylated cholesterol metabolites, to 27-oxygenated steroids (Fig. 1). Sterol 27-hydroxylase is capable of hydroxylating the C27-methyl group more than once, leading to a C27-carboxylic group6
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growth hormone. Endocrinology, 134, 990–997 39 Childs, G.V. et al. (1994) Cytochemical detection of GnRH binding sites on rat pituitary cells with LH, FSH and GH antigens during diestrous upregulation. Endocrinology 134, 1943–1951 40 Childs, G.V. et al. (1997) Differential effects of inhibin on gonadotropin stores and gonadotropin releasing hormone binding to pituitary cells from cycling female rats. Endocrinology 138, 1577–1584 41 Childs, G.V. and Unabia, G. (1997) Cytochemical studies of the effects of activin on gonadotropin releasing hormone (GnRH) binding by pituitary gonadotropes and
The Mammalian Fatty Acid-binding Protein Multigene Family: Molecular and Genetic Insights into Function Ann Vogel Hertzel and David A. Bernlohr
Intracellular fatty acid-binding proteins associate with fatty acids and other hydrophobic biomolecules in an internal cavity, providing for solubilization and metabolic trafficking. Analyses of their in vivo function by molecular and genetic techniques reveal specific function(s) that fatty acid-binding proteins perform with respect to fatty acid uptake, oxidation and overall metabolic homeostasis. Intracellular fatty acid-binding proteins (FABPs) are members of a multigene family encoding ~15-kDa proteins, which bind a hydrophobic ligand in a non-covalent, reversible manner (reviewed in Refs 1–3). The nine family members have between 20% and 70% identity in their amino acid sequence. Despite the wide variance in primary sequence, numerous X-ray crystal structures have shown a common tertiary fold forming a b-barrel (Fig. 1). The barrel comprises ten antiparallel bstrands, linked by hydrogen bonds, which are organized into two nearly orthogonal b-sheets. Importantly, the A. Vogel Hertzel and D.A. Bernlohr are at the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Avenue, St Paul, MN 55108, USA. Tel: 11 612 624 2712, Fax: 11 612 625 5780, email: [email protected]. umn.edu
TEM Vol. 11, No. 5, 2000
b-sheets create an internal, water-filled cavity, lined with ~50% polar amino acids. Although the cavity is significantly larger (two-three times) than the
growth hormone cells. J. Histochem. Cytochem. 45, 1603–1610 42 Childs, G.V. et al. (1999) Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats. J. Endocrinol. 162, 177–187 43 Childs, G.V. et al. (2000) Differential expression of growth hormone mRNA by somatotropes and gonadotropes in male and cycling female rats. Endocrinology 141, 1560–1570 44 Lee, B.L. et al. (1993) Expression of follistatin mRNA in somatotropes and mammotropes early in the estrous cycle. J. Histochem. Cytochem. 41, 955–960
volume of a fatty acid, typically only a single fatty acid is bound in the cavity, with the carboxylate group oriented inwards, coordinated by a tyrosine and two arginine residues. At the N-terminus of FABPs, a helix–loop–helix motif forms a cap-like structure on the ‘top’ of the barrel. Because there is no obvious opening for the fatty acids to enter or exit the cavity, the portal hypothesis was proposed. This involves a transient conformational change around the helix–loop–helix area and adjacent loops connecting b-strands, thereby allowing the fatty acid to enter or exit the cavity4 (Fig. 1). The function(s) of these proteins within cells has remained elusive. Although the in vitro binding of a fatty acid has been analyzed extensively, the in vivo function is less well defined. It has been hypothesized that owing to the low solubility of fatty acids in an
Figure 1. Ribbon diagram of the X-ray crystal structure of fatty acid-binding protein 4 (FABP4). (a) Oleate ligand (yellow) bound in the cavity of FABP4 with the helix–loop–helix motif in top part of the figure. b-Strands A–E are in front and b-strands F–J are behind. (b) ‘Top’ view of FABP4, looking down on the portal region. The portal is surrounded by the second a-helix, and loops (noted with arrows) between b-strands C and D, and b-strands E and F. This figure was created with the use of RasMol v2.6 with data from the Brookhaven Protein DataBank, Id# 1LID.
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aqueous environment the FABPs are required to facilitate their solubilization. Additional proposed functions include facilitating the influx of fatty acids across the plasma membrane (PM) (preventing efflux by intracellular association), transport of the fatty acids within the cell, determining compartmentalization for storage, modulation of activity of enzymes involved in fatty acid metabolism and protection of enzymes and membranes from detergent-like effects of fatty acids. The question that arises then is: why are there nine different members seemingly performing the same function? A potential clue lies in the unique tissue-specific expression patterns of each member of the family (Table 1), implying that the proteins might be carrying out specialized functions, thereby justifying the need for a variety of family members. The use of molecular and genetic methods to analyze FABP functions has, in some cases, shed light upon the functional aspects of some FABPs. Because many of these proteins have been cloned from various organisms, and from different cell or tissue types, each has been named according to the location studied. Owing to the many names for the same or orthologous proteins, we will refer to each FABP only by its gene name (Table 1). • FABP1 FABP1 is found in liver, intestine and kidney and is unique in that it can bind two ligands, the first in the more typical configuration of carboxyl group inward and the second in the opposite orientation, with the carboxyl group outward5. In addition to binding long-chain fatty acids, it binds fatty acyl-CoAs, peroxisome proliferators, prostaglandins, bile acids, bilirubin, hydroxy and hydroperoxy metabolites of fatty acids, lysophosphatidic acids, selenium, heme and other hydrophobic ligands6. Transfection of Fabp1 into fibroblasts stimulated the initial rate and extent of fatty acid uptake and increased esterification into specific lipid pools7,8. In HepG2 cells, peroxisome proliferators increased FABP1 expression as well as stimulating fatty acid uptake. Stable transfection of antisense
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Table 1. The fatty acid-binding protein multigene familya Gene name
Common name
Other names (species)
FABP1
Liver
FABP2
Intestinal
FABP3
Heart/muscle
FABP4
Adipocyte
FABP5
Epidermal
FABP6
Ileal
FABP7
Brain
FABP9
Testis
MP2
Myelin P2
L-FABP h-FABP Z protein I-FABP gFABP H-FABP M-FABP MDGI c-FABP ALBP aP2 A-FABP p422 p15 Klbp (murine) Mal-1 (murine) E-FABP (human) PA-FABP (human) C-Fabp (rat) S-Fabp (rat) Le-Lbp (rat) Da11 (rat) LP2 (bovine) Melanogenic inhibitor IlLBP ILBP BABP IL-FABP I15P Gastrotropin B-FABP BLBP MRG R-FABP (chicken) T-FABP TLBP PERF15 Myelin P2 PMP2 MLBP My-FABP
Expression Liver, intestine, kidney, lung(est)
Intestine Heart, mammary, skeletal muscle
Adipose tissue, macrophages
Abnormal skin, lens, adipose tissue, endothelial cells, lung, mammary cells, brain, stomach, tongue, stomach, placenta, heart, skeletal muscle, intestine, testis, retina
Ileum
Brain, olfactory bulb
Testis
Schwann cells
a Primary references for this table and explanations for all names given above can be found at the fatty acidbinding protein web site: http://cbs.umn.edu/bmbb/FABP.html
RNA reduced FABP1 expression by 84% and reduced the rate of oleate uptake by 66% (Ref. 9). Consistent with a role for FABP1 in uptake, growth-hormone injections of rats increased FABP1 levels and fatty acid uptake, whereas hypophysectomized rats had a decrease in FABP1 levels, which also correlated with a decrease in fatty acid uptake10–12.
• FABP2 FABP2 has been studied intensively since the initial reporting that the A54T polymorphism in FABP2 alleles of Pima Indians was linked to a predisposition to type 2 diabetes13. This single amino acid substitution doubles the affinity of FABP2 for fatty acids and increases fatty acid uptake14. There is a general TEM Vol. 11, No. 5, 2000
correlation of FABP2 A54T with bodymass index (BMI), fasting triglycerides, high basal insulin levels, but not diabetes mellitus in obese Japanese15–17, aboriginal Canadians18 and Guadeloupe Indian populations19. In subsequent studies of Pima Indians, A54T was not associated with insulin resistance, but with a high lipid oxidation rate and raised high-density lipoprotein (HDL) and low-density lipoprotein (LDL) triglycerides20. Several systems were used to correlate FABP2 levels with fatty acid uptake. Fabp2 transfected into mouse fibroblasts or differentiated intestinal cells did not increase fatty acid uptake, but increased esterification into specific lipid pools7,21,22. Undifferentiated mouse pluripotent embryonic stem cells transfected with Fabp2 increased fatty acid uptake 1.7-fold and increased the intracellular diffusion constant similarly. Differentiation of these embryonic stem cells into fibroblast-like cells resulted in reduced FABP2 levels and decreased fatty acid uptake23. Furthermore, in a human enterocyte cell line, Caco-2, treatment with epidermal growth factor (EGF) decreases levels of FABP2 and inhibits fatty acid uptake24. Therefore, in all of these cases, the levels of FABP2 correlated with the ability of various cell lines to take up fatty acids. • FABP3 The heart typically derives 70% of its energy requirements from the oxidation of fat, even more upon fasting or when in a diabetic state. Owing to the high demand for lipids in the heart, FABP3 has been proposed to play a role in transporting fatty acids from the sarcolemma to their intracellular sites of metabolism. Not only has FABP3 been shown to be expressed abundantly in cardiomyocytes, and at lower levels in other heart cells, but it is also expressed in red skeletal muscles, renal cortex, testis and brain25. FABP3 levels are influenced by testosterone (increased by 33%), endurance training (increased by 29%) and the circadian rhythm (increased by 100% in dark over light). Changes are also seen in pathophysiological conditions; those that tend to increase fatty acid oxidation increase TEM Vol. 11, No. 5, 2000
FABP3 (Ref. 25). In an attempt to define the role of FABP3 in lipid metabolism more fully, transfection of cell lines that do not contain FABP3 has yielded mixed results. In 3T3 pre-adipocytes and COS7 cells, transfection of Fabp3 cDNA did not alter fatty acid uptake26. However, in a human breast cancer line, MCF7, transient expression of bovine Fabp3 increased uptake of oleate 67%. The increase in uptake might be underestimated owing to the presence of FABP5 in MCF7 cells27. To test the function of FABP3 in vivo, a disruption of the Fabp3 locus in 129 BALB/c mice was created28. These mice are fertile and have no obvious phenotypic abnormalities. Histological sections of heart, skeletal muscle and liver tissues from three-month old mice appeared normal. Fatty acid influx experiments showed the first differences: cardiac uptake of long-chain fatty acids in Fabp3 null mice was dramatically decreased compared with wild-type mice. The specificity in the defect of fatty acid uptake was tested by comparing the initial rate of uptake for a fatty acid bound by FABP3 (palmitate) to that of a non-ligand (octanoate). The rate of palmitate uptake was reduced by 45% in cardiac myocytes of FABP3-deficient mice; in contrast, uptake of octanoate was unaltered. Owing to the inability to internalize and perhaps transport fatty acids efficiently, the heart muscle switches to glucose utilization as an energy source, as confirmed by the dramatic increase in glucose transport (Fig. 2)28. Further analysis of the heart demonstrated no change in the levels of putative fatty acid transport proteins, acyl-CoA synthetase, or acyl-CoA-binding protein. The molecular defect appears to be before the b-oxidation step because the fatty acid oxidative capacities of heart homogenates were unaltered if the fatty acids were delivered on albumin29. In resting mice, this change in energy source appeared to be tolerated. However, when FABP3 null mice were stressed with the higher energy demands of exercise, these mice exhibited exercise intolerance, as indicated by higher levels of exhaustion and even
death. As such, FABP3 appears to be required for efficient cardiac fatty acid transport and oxidation28. • FABP4 To investigate the in vivo function of FABP4, a targeted disruption of the locus was made in C57BL/6J mice30. These mice appeared healthy and seemed to develop adipocytes normally. The lack of a phenotypic change prompted a search for the expression of genes encoding other FABPs in adipocytes. Another member of the FABP gene family, Fabp5, was upregulated in adipocytes 20–40-fold (mRNA), with the protein being increased 13fold30,31. When the mice were stressed with diet-induced obesity, fasting plasma glucose, insulin and cholesterol levels were decreased relative to wildtype mice, even though the mice gained a similar amount of weight. The serum triglycerides in obese wild-type mice were significantly raised relative to obese FABP4 null mice, whose serum triglycerides remained low, potentially because of an alteration in triglyceride synthesis and/or secretion. Glucose and insulin tolerance tests indicated the maintenance of insulin sensitivity in obese FABP4 null mice, contrasting
FABP3 +/+ G
FABP3 –/–
FA FA
G
G
FA PM
G
FA FABP3
β-Oxidation Fuel: FA
G G
G G
G G
G
Glycolysis Fuel: G
trends in Endocrinology and Metabolism
Figure 2. Schematic comparison of cardiac cells from wild-type (Fabp31/1) and Fabp32/2 null mice, demonstrating the change in energy source. Because they cannot properly influx and oxidize FAs, FABP3 null mice switch to glucose utilization replacing the normal use of fatty acids. Abbreviations: FA, fatty acid; FABP3, fatty acid-binding protein 3; G, glucose molecule; PM, plasma membrane.
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FA FA PM Uptake
Esterification? Metabolism?
FABP4
Lipolysis
FA FABP4
Insulin resistance?
HSL Triglyceride droplet FA
trends in Endocrinology and Metabolism
Figure 3. Schematic model of intracellular transport of FAs by FABP4. Functions include binding of FAs, interaction with HSL, the delivery of FAs for esterification and/or other metabolism, release of FAs from lipolysis and providing a link to insulin resistance. Abbreviations: FA, fatty acid; FABP4, fatty acid-binding protein 4; HSL, hormone-sensitive lipase; PM, plasma membrane.
with the insulin resistance that occurred in the wild-type mice. Thus, a single disruption in FABP4 created a genetic model to unlink obesity and insulin resistance. Changes in lipid metabolism were manifested as a threefold increase in the total amount of free fatty acids in adipocytes. Consistent with this, a decrease of ~40% in basal and stimulated lipolysis was also noted, without a change in the production of hormone-sensitive lipase or perilipin. The composition of the fatty acids in the adipocyte remained unaltered, whereas there were a few small but significant differences in the fatty acid composition of the sera30,31. Another indication of the function of FABP4 came from the finding that FABP4 interacts directly with hormonesensitive lipase32 (Fig. 3). Originally identified through yeast two-hybrid assays, the interaction was confirmed with other experiments testing protein– protein interactions, including glutathione S-transferase (GST)-pulldowns and co-immunoprecipitations of the hormone-sensitive lipase (HSL)–FABP4
178
complex. The interaction domain of HSL resides in the N-terminal portion of the protein, whereas the catalytic domain has been localized to the C-terminus. Although several interpretations are possible, this interaction is consistent with a role for FABP4 in facilitating fatty acid trafficking in response to lipolytic stimulation. These results suggest FABP4 functions to traffic fatty acids away from the triglyceride after hydrolysis. It is still unclear exactly where FABP4 is taking the fatty acid: to the PM for efflux or to internal membranes for re-esterification? • FABP5 Several types of cells or tissues have been reported to express the epidermal Fabp5 (Table 1), including lens, adipose, mammary and endothelial cells, stratified epithelia of epidermis and tongue, stomach, heart, brain, liver, spleen, muscle, lung, intestine, bone marrow, renal medulla, testis, urothelium and retina33,34. Binding studies have been used to elucidate ligands for FABP5; these include long-chain fatty acids but do not include retinoic acid2,34,35. FABP5 has been shown to be upregulated in a number of altered states with disturbed lipid profiles. It is highly upregulated in benign papillomas, carcinomas36, skin warts and psoriatic keratinocytes (an increase of approximately 80-fold)35,37. In addition, it is upregulated in bladder transitional-cell carcinomas38 and peripheral nerve trauma39. Increased Fabp5 expression is seen in the hippocampal glia of the brain after systemic treatment with the seizurecausing chemical kainic acid40. In addition, sodium dodecyl sulfate (SDS) or acetone treatment of human skin, known to disturb the barrier function as seen by transepidermal water loss, upregulates FABP5 production41–43. Independent of development or differentiation, this FABP exhibits the most highly regulated synthesis of all FABP forms. However, the specific function(s) for FABP5 in the various cell types remains undefined. • FABP6 FABP6 is found in the ileum, ovary and adrenal gland44,45, and although it is homologous to the other members of the
family, it has low or no binding affinity for long-chain fatty acids, but has high affinity for bile acids. It has been hypothesized to function as a cytosolic receptor for bile acids transported by the sodiumdependent action of the ileal bile-acid transporter46. Recently, bile acids have been shown to regulate the expression of Fabp6 (Ref. 47) and this regulation is achieved through the actions of the farnesoid-X-receptor (FXR), an orphan receptor whose physiological ligands are bile acids48. • FABP7 Fabp7 is expressed at the highest levels in the developing brain and retina, with decreased levels in the adult49. FABP7 binds very-long-chain fatty acids, such as docosahexaenoic acid with very high affinity, ~20-fold greater than any longchain fatty acid50. Of these, it will bind the longer C18–C20, but not C16 fatty acids, and seems to prefer unsaturated to saturated fatty acids. In addition, it has been shown to bind oleoyl-CoA as well as lysophosphotidic acid51. The most probable in vivo ligand is docosahexaenoic acid, because it is bound with high affinity and the requirement for docosahexaenoic acid for proper brain development corresponds to the same time period in which Fabp7 is expressed50. FABP7 might specifically solubilize the very-long-chain fatty acids, thereby allowing for proper uptake and intracellular trafficking. • Myelin P2 P2 is expressed in Schwann cells and is one of the major proteins of peripheral myelin. P2 avidly associates with membrane phospholipids, and because 70% of myelin is composed of lipids, it is postulated that P2 is a lipid carrier involved in the production and maintenance of myelin. In vitro binding studies indicated high affinity for oleic acid, retinoic acid and retinol52. Purification of P2 from myelin identified the relative profile of myelin lipids bound by P2 to be similar to that present in myelin, with some alterations in the relative ratios53. • Conclusion In 1987, Sweetser et al.54 wrote that FABPs were ‘abundant proteins in search TEM Vol. 11, No. 5, 2000
of a function’. The advent of the knockout mouse and other molecular technologies has strongly implicated FABPs as more than simple cellular fatty acid buffers; they are now thought to be active fatty acid chaperones. The proteins not only protect and shuttle fatty acids within the cell, but actively participate in the acquisition or removal of fatty acids from intracellular sites, contributing specificity through selection in ligand binding. Because transgenic and knockout studies have provided the most informative results indicating potential role(s) of FABPs in lipid metabolism, the development of additional null lines will probably provide further insights into the functions of these ubiquitous fatty acid chaperones. • Acknowledgements We thank members of the Bernlohr laboratory for helpful suggestions and comments during the preparation of this manuscript. This work was supported by grants NSF 9816575, NIH DK 53189 and NRSA 1 F32 DK09599-01. References 1 Glatz, J.F. and van der Vusse, G.J. (1996) Cellular fatty acid-binding proteins: their function and physiological significance. Prog. Lipid Res. 35, 243–282 2 Bernlohr, D.A. et al. (1997) Intracellular lipid-binding proteins and their genes. Annu. Rev. Nutr. 17, 277–303 3 Coe, N.R. and Bernlohr, D.A. (1998) Physiological properties and functions of intracellular fatty acid-binding proteins. Biochim. Biophys. Acta 1391, 287–306 4 Sacchettini, J.C. et al. (1989) Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate. J. Mol. Biol. 208, 327–339 5 Thompson, J. et al. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates. J. Biol. Chem. 272, 7140–7150 6 Keler, T. et al. (1997) Liver fatty acid binding protein and mitogenesis in transfected hepatoma cells. Adv. Exp. Med. Biol. 400A, 517–524 7 Prows, D.R. et al. (1995) Intestinal and liver fatty acid-binding proteins differentially affect fatty acid uptake and esterification in L-cells. Lipids 30, 907–910 8 Murphy, E.J. et al. (1996) Liver fatty acidbinding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism. Biochim. Biophys. Acta 1301, 191–198 9 Wolfrum, C. et al. (1999) Variation of livertype fatty acid-binding protein content in the human hepatoma cell line HepG2 by TEM Vol. 11, No. 5, 2000
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correlates with the co-expression of an integral 93-kDa and a peripheral 14-kDa bile acid-binding membrane protein along the duodenum–ileum axis. J. Biol. Chem. 268, 18035–18046 47 Kanda, T. et al. (1998) Regulation of expression of human intestinal bile acid-binding protein in Caco-2 cells. Biochem. J. 330, 261–265 48 Grober, J. et al. (1999) Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene – involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer. J. Biol. Chem. 274, 29749–29754 49 Shimizu, F. et al. (1997) Isolation and expression of a cDNA for human brain fatty acidbinding protein (B-FABP). Biochim. Biophys. Acta 1354, 24–28
Sterol 27-hydroxylase Deficiency: A Rare Cause of Xanthomas in Normocholesterolemic Humans Ingemar Björkhem and Eran Leitersdorf
Cerebrotendinous xanthomatosis is characterized by the accumulation of cholestanol and cholesterol in xanthomas and brain causing a number of severe symptoms. More than 20 different mutations have been identified in the gene encoding sterol 27-hydroxylase. Defects in the gene lead to reduced bile acid biosynthesis, with accumulation of 7a-hydroxylated intermediates, one of which is a precursor to cholestanol. The disease can be treated successfully with chenodeoxycholic acid, which reduces the upregulation of cholesterol 7a-hydroxylase and, therefore, the formation of cholestanol. Disruption of the gene encoding sterol 27-hydroxylase in mice does not have the same metabolic consequences as in humans.
Familial hypercholesterolemia is by far the most common cause of xanthomas. However, among patients with xanthomas, there are two small subgroups in which increased levels of cholesterol in the circulation are not the primary I. Björkhem is at the Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden; and E. Leitersdorf is at the Department of Medicine, Hadassah University Hospital, IL-91120 Jerusalem, Israel. Tel: 146 8 585 812 35, Fax: 146 8 585 812 60, email: [email protected]
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cause for xanthomas development. These two rare sterol storage diseases are cerebrotendinous xanthomatosis (CTX) and phytosterolemia. CTX has now been characterized at the gene level and is reviewed here. For a more extensive review, see Ref. 1. • Phenotype CTX was first described by Van Bogaert et al. in 1937 (Ref. 2). The patient they described suffered from dementia, ataxia, cataracts and xanthomas in the tendons and nervous system. Several hundred
50 Xu, L.Z. et al. (1996) Ligand specificity of brain lipid-binding protein. J. Biol. Chem. 271, 24711–24719 51 Myers-Payne, S.C. et al. (1996) Isolation and characterization of two fatty acid binding proteins from mouse brain. J. Neurochem. 66, 1648–1656 52 Uyemura, K. et al. (1984) Lipid binding activities of the P2 protein in peripheral nerve myelin. Neurochem. Res. 9, 1509–1514 53 Riccio, P. et al. (1998) Purification of bovine P2 myelin protein with bound lipids. NeuroReport 9, 2769–2773 54 Sweetser, D.A. et al. (1987) The metabolic significance of mammalian fatty-acidbinding proteins: abundant proteins in search of a function. Annu. Rev. Nutr. 7, 337–359
patients have since been described, with most cases being in Japan, Israel and The Netherlands (so far, no cases have been diagnosed in Australia). In CTX patients, cholesterol levels were raised in the tissues but not in the blood, and the xanthomas were shown to contain high levels of cholestanol, the 5a-saturated analog of cholesterol3. The major clinical manifestations of CTX are caused by the accumulation of cholestanol and cholesterol in almost every tissue, including the central nervous system (CNS). • Characterization of CTX and the Role of the Defective Enzyme In 1974, Setoguchi et al. made the key discovery that CTX patients have a defect in bile-acid biosynthesis, with incomplete oxidation of the C27-steroid side chain4. In 1980, Oftebro et al. reported that a liver biopsy from a patient with CTX had an almost complete lack of mitochondrial sterol 27-hydroxylase5. Sterol 27-hydroxylase is a mitochondrial cytochrome P-450 species (CYP27) that is essential for the normal degradation of the steroid side chain in connection with formation of bile acids from cholesterol. Sterol 27-hydroxylase catalyzes the first step in the normal oxidation of the steroid side chain, converting cholesterol, as well as different 7a-hydroxylated cholesterol metabolites, to 27-oxygenated steroids (Fig. 1). Sterol 27-hydroxylase is capable of hydroxylating the C27-methyl group more than once, leading to a C27-carboxylic group6
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TEM Vol. 11, No. 5, 2000