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PubMed1998

Peroxisomal D-hydroxyacyl-CoA dehydrogenase deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein deficiency.

Peroxisomes play an essential role in a number of different metabolic pathways, including the beta-oxidation of a distinct set of fatty acids and fatty acid derivatives. The importance of the peroxisomal beta-oxidation system in humans is made apparent by the existence of a group of inherited diseases in which peroxisomal beta-oxidation is impaired. This includes X-linked adrenoleukodystrophy and other disorders with a defined defect. On the other hand, many patients have been described with a defect in peroxisomal beta-oxidation of unknown etiology. Resolution of the defects in these patients requires the elucidation of the enzymatic organization of the peroxisomal beta-oxidation system. Importantly, a new peroxisomal beta-oxidation enzyme was recently described called D-bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity primarily reacting with alpha-methyl fatty acids like pristanic acid and di- and trihydroxycholestanoic acid. In this patient we describe the first case of D-bifunctional protein deficiency as resolved by enzyme activity measurements and mutation analysis. The mutation found (Gly16Ser) is in the dehydrogenase coding part of the gene in an important loop of the Rossman fold forming the NAD+-binding site. The results show that the newly identified D-bifunctional protein plays an essential role in the peroxisomal beta-oxidation pathway that cannot be compensated for by the L-specific bifunctional protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha).

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the steroid/nuclear receptor superfamily and mediates the biological and toxicological effects of peroxisome proliferators. To determine the physiological role of PPARalpha in fatty acid metabolism, levels of peroxisomal and mitochondrial fatty acid metabolizing enzymes were determined in the PPARalpha null mouse. Constitutive liver beta-oxidation of the long chain fatty acid, palmitic acid, was lower in the PPARalpha null mice as compared with wild type mice, indicating defective mitochondrial fatty acid catabolism. In contrast, constitutive oxidation of the very long chain fatty acid, lignoceric acid, was not different between wild type and PPARalpha null mice, suggesting that constitutive expression of enzymes involved in peroxisomal beta-oxidation is independent of PPARalpha. Indeed, the PPARalpha null mice had normal levels of the peroxisomal acyl-CoA oxidase, bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase), and thiolase but lower constitutive expression of the D-type bifunctional protein (hydratase + 3-hydroxyacyl-CoA dehydrogenase). Several mitochondrial fatty acid metabolizing enzymes including very long chain acyl-CoA dehydrogenase, long chain acyl-CoA dehydrogenase, short chain-specific 3-ketoacyl-CoA thiolase, and long chain acyl-CoA synthetase are also expressed at lower levels in the untreated PPARalpha null mice, whereas other fatty acid metabolizing enzymes were not different between the untreated null mice and wild type mice. A lower constitutive expression of mRNAs encoding these enzymes was also found, suggesting that the effect was due to altered gene expression. In wild type mice, both peroxisomal and mitochondrial enzymes were induced by the peroxisome proliferator Wy-14,643; induction was not observed in the PPARalpha null animals. These data indicate that PPARalpha modulates constitutive expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid beta-oxidation, thus establishing a role for the receptor in fatty acid homeostasis.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder.

Peroxisomal beta-oxidation proceeds from enoyl-CoA through D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA by the D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxy-acyl-CoA dehydrogenase bifunctional protein (d-bifunctional protein), and the oxidation of bile-acid precursors also has been suggested as being catalyzed by the d-bifunctional protein. Because of the important roles of this protein, we reinvestigated two Japanese patients previously diagnosed as having enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein) deficiency, in complementation studies. We found that both the protein and the enzyme activity of the d-bifunctional protein were hardly detectable in these patients but that the active L-bifunctional protein was present. The mRNA level in patient 1 was very low, and, for patient 2, mRNA was of a smaller size. Sequencing analysis of the cDNA revealed a 52-bp deletion in patient 1 and a 237-bp deletion in patient 2. This seems to be the first report of D-bifunctional protein deficiency. Patients previously diagnosed as cases of L-bifunctional protein deficiency probably should be reexamined for a possible d-bifunctional protein deficiency.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Peroxisomal bifunctional protein deficiency revisited: resolution of its true enzymatic and molecular basis.

In the past few years, many patients have been described who have a defect of unknown origin in the peroxisomal beta-oxidation pathway. Complementation analysis has been done by various groups to establish the extent of the genetic heterogeneity among the patients. These studies were based on the use of two established cell lines, one with a deficiency of acyl-CoA oxidase and one with a deficiency of l-bifunctional protein (l-BP), and they showed that most patients belong to the l-BP-deficient group. However, molecular analysis of the cDNA encoding l-BP in patients failed to show any mutations. The recent identification of a new d-specific bifunctional protein (d-BP) prompted us to reinvestigate the original patient with presumed l-BP deficiency. In a collaborative effort, we have now found that the true defect in this patient is at the level of the d-BP and not at the level of the l-BP. Our results suggest that most, if not all, patients whose condition has been diagnosed as l-BP are, in fact, d-BP deficient. We tested this hypothesis in nine patients whose condition was diagnosed as l-BP deficiency on the basis of complementation analysis and found clear-cut mutations in the d-BP cDNA from all patients.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency.

D-bifunctional protein is involved in the peroxisomal beta-oxidation of very long chain fatty acids, branched chain fatty acids and bile acid intermediates. In line with the central role of D-bifunctional protein in the beta-oxidation of these three types of fatty acids, all patients with D-bifunctional protein deficiency so far reported in the literature show elevated levels of very long chain fatty acids, branched chain fatty acids and bile acid inter-mediates. In contrast, we now report two novel patients with D-bifunctional protein deficiency who both have normal levels of bile acid intermediates. Complementation analysis and D-bifunctional protein activity measurements revealed that both patients had an isolated defect in the enoyl-CoA hydratase domain of D-bifunctional protein. Subsequent mutation analysis showed that both patients are homozygous for a missense mutation (N457Y), which is located in the enoyl-CoA hydratase coding part of the D-bifunctional protein gene. Expression of the mutant protein in the yeast Saccharomyces cerevisiae confirmed that the N457Y mutation is the disease-causing mutation. Immunoblot analysis of patient fibroblast homogenates showed that the protein levels of full-length D-bifunctional protein were strongly reduced while the enoyl-CoA hydratase component produced after processing within the peroxisome was undetectable, which indicates that the mutation leads to an unstable protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Purification and properties of rat D-3-hydroxyacyl-CoA dehydratase: D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

We have previously purified two D-3-hydroxyacyl-CoA dehydratase preparations from human liver. One preparation contained a 77-kDa polypeptide and smaller polypeptides, and the other was a homodimer of a 46-kDa polypeptide. Three different purified rat peroxisomal D-3-hydroxyacyl-CoA dehydratase preparations have been reported. Therefore, rat enzyme was purified in this study to confirm the enzyme structure. Two preparations with similar molecular structures to the human enzyme preparations were obtained, and these were similar to each other in immunochemical and catalytic properties. It was suggested that the native enzyme was a homodimer of the 77-kDa polypeptide, and this enzyme was modified to a homodimer of the 46-kDa polypeptide, because conversion of the 77-kDa polypeptide to smaller polypeptides including the 46-kDa polypeptide was clearly observed during purification. Rat liver subcellular fractionation study indicates that this enzyme is located in peroxisomes. The enzyme preparation containing the 77-kDa polypeptide catalyzed the D-3-hydroxyacyl-CoA dehydrogenase reaction as well as the dehydratase reaction. Thus, it is proposed that this enzyme is D-3-hydroxyacyl-CoA dehydratase/ D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Structure of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

When D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein was purified from human liver, two preparations were obtained. One contained a 77-kDa polypeptides as the main and minor smaller polypeptides including a 46-kDa polypeptide, and this preparation showed both the dehydratase and dehydrogenase activities. The other preparation was a homodimer of the 46-kDa polypeptide and showed only the dehydratase activity. Further analysis indicated that the native enzyme is a homodimer of 77-kDa polypeptide, which was proteolytically modified during purification. The cDNA for the human 77-kDa polypeptide was cloned. The amino acid sequences of the peptides derived from the components of the enzyme preparations were located in the deduced amino acid sequence of the cDNA. The preparation containing the 77-kDa polypeptide was treated with a protease, and two monofunctional fragments were separated. The dehydrogenase and dehydratase fragments were located on the amino- and carboxyl-terminal sides, respectively, of the deduced amino acid sequence of the cDNA. The protein expressed by the cDNA with the entire coding region exhibited both the dehydratase and dehydrogenase activities, and that expressed by a truncated version covering the carboxyl-terminal side exhibited only the dehydratase activity. The cloned cDNA was identical to the human 17 beta-hydroxysteroid dehydrogenase IV cDNA.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Physiological role of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

The second and third reactions of the peroxisomal beta-oxidation spiral are thought to be catalyzed by enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein). Recently, we found the presence of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) in mammalian peroxisomes. Therefore, we studied the physiological role of the D-bifunctional protein. The contents of the L- and D-bifunctional proteins were about 0.01 and 0.5 microg/mg protein, respectively, in cultured human skin fibroblasts. The activity of conversion of hexadecenoyl-CoA to 3-ketopalmitoyl-CoA by the D-bifunctional protein was estimated to be about 0.5 milliunit/mg of fibroblast protein. This value was about 100-fold that of the L-bifunctional protein in the fibroblasts. From comparison of the activities of the bifunctional proteins with the rate of palmitate oxidation and the activities of acyl-CoA oxidase and 3-ketoacyl-CoA thiolase, it is proposed that the D-bifunctional protein plays a major role in the peroxisomal oxidation of palmitate in the fibroblasts. The contents of both the L- and D-bifunctional proteins in liver were about 2.5 microg/mg protein. Therefore, it is suggested that the D-bifunctional protein also plays a significant role in human liver peroxisomal fatty acid oxidation. Actions of the bifunctional proteins on enoyl forms of other acyl-CoA derivatives were examined. The D-bifunctional protein but not the L-bifunctional protein reacted with 2-methylhexadecenoyl-CoA and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-enoyl-CoA. We propose that, among the reactions of the distinct group of carboxylates oxidized specifically in peroxisomes, oxidation of 2-methyl-branched fatty acids and side-chain shortening of cholesterol for bile acid formation are catalyzed by the D-bifunctional protein, but not the L-bifunctional protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Bile acid profiles in a peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency.

Bile acid profiles in serum, urine and bile from an infant with a peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-bifunctional protein) deficiency were analyzed by means of gas-liquid chromatography, gas-liquid chromatography-mass spectrometry, and high-performance liquid chromatography. As in such several peroxisomal disorders as Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, the accumulation of C27-bile acid intermediates was also demonstrated in the infant with D-bifunctional protein deficiency, accounting for 74% of the total bile acids in serum, 59% in urine, and 35% in bile. In addition, the major constituents of the C27-bile acids were (24R,25R)- and (24R,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5be ta-cholestanoic acids along with small amounts of their 24S counterparts. Since immunoreactive acyl-CoA oxidase, L-bifunctional protein, and thiolase were all present in the liver, the impairment of the oxidative side-chain cleavage in bile acid biosynthesis is considered to be due to the defect of D-bifunctional protein.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Thiolase involved in bile acid formation.

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Stereospecific formation of (24R,25R)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid catalyzed with a peroxisomal bifunctional D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase.

The absolute configuration of 3 alpha,7 alpha,12 alpha, 24-tetrahydroxy-5 beta-cholestan-26-oic acid CoA ester (V-CoA) produced by the incubation of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid CoA ester (24E-THC-CoA) with D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-bifunctional protein) was investigated. When 24E-THC-CoA was incubated with D-bifunctional protein the formation of only one isomer (24R,25R-isomer) of four possible stereoisomers of V-CoA was observed, which suggested the cis-addition of water to a side chain double bond of 24E-THC-CoA. The dehydration reaction of V-CoA catalyzed by D-bifunctional protein was also observed when (24R,25R)-V-CoA was used as a substrate. The other three isomers (24R,25S-, 24S,25R- and 24S,25S-isomers) were not dehydrated with D-bifunctional protein. These results showed that D-bifunctional protein catalyzes stereospecifically the hydration and dehydration step in bile acid biosynthesis.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

17Beta-hydroxysteroid dehydrogenases in human bone cells.

Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17beta-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17beta-HSD I was detectable only in MG63 cells, albeit at low levels, while 17beta-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17beta-HSD III was absent from each bone cell type. 17Beta-HSD IV was the only isoform present in all bone cells analyzed. Further analysis of the expression of 17beta-HSD IV in these bone cells by immunoblotting revealed both the full-length 83 kDa protein and the proteolytic 38 kDa form. The kinetic parameters for estradiol oxidation by purified recombinant 17beta-HSD IV (Km = 49.7 microM, Vmax = 79.4 nmol/minute/mg of protein) and its HSD-domain (Km = 79.4 microM, Vmax = 476 nmol/minute/mg of protein) were significantly higher than previously reported, but consistent with the values obtained with crude cell-free extracts of SaOS-2 cells (Km = 98.8 microM, Vmax = 0.07 nmol/minute/mg of protein) which contain only 17beta-HSD IV based on RT-PCR. These studies show that bone cells have the capacity to interconvert circulating estrogens and suggest that bone cell 17beta-HSDs serve primarily to attenuate the continuing actions of estradiol through conversion to its less potent form, estrone, under certain conditions.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Molecular changes in the D-bifunctional protein cDNA sequence in Australasian patients belonging to the bifunctional protein complementation group.

The cDNA sequence for the human D-bifunctional protein (D-BP: 17 beta-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209-537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3-349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1-302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the beta-oxidation defect in patients belonging to the BP CG.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Unique multifunctional HSD17B4 gene product: 17beta-hydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase involved in Zellweger syndrome.

Six types of human 17beta-hydroxysteroid dehydrogenases catalyzing the conversion of estrogens and androgens at position C17 have been identified so far. The peroxisomal 17beta-hydroxysteroid dehydrogenase type 4 (17beta-HSD 4, gene name HSD17B4) catalyzes the oxidation of estradiol with high preference over the reduction of estrone. The highest levels of 17beta-HSD 4 mRNA transcription and specific activity are found in liver and kidney followed by ovary and testes. A 3 kb mRNA codes for an 80 kDa (737 amino acids) protein featuring domains which are not present in the other 17beta-HSDs. The N-terminal domain of 17beta-HSD 4 reveals only 25% amino acid similarity with the other types of 17beta-HSDs. The 80 kDa protein is N-terminally cleaved to a 32 kDa enzymatically active fragment. Both the 80 kDa and the N-terminal 32 kDa (amino acids 1-323) protein are able to perform the dehydrogenase reaction not only with steroids at the C17 position but also with D-3-hydroxyacyl-coenzyme A (CoA). The enzyme is not active with L-stereoisomers. The central part of the 80 kDa protein (amino acids 324-596) catalyzes the 2-enoyl-acyl-CoA hydratase reaction with high efficiency. The C-terminal part of the 80 kDa protein (amino acids 597-737) facilitates the transfer of 7-dehydrocholesterol and phosphatidylcholine between membranes in vitro. The HSD17B4 gene is stimulated by progesterone, and ligands of PPARalpha (peroxisomal proliferator activated receptor alpha) such as clofibrate, and is down-regulated by phorbol esters. Mutations in the HSD17B4 lead to a fatal form of Zellweger syndrome.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Local estradiol metabolism in osteoblast- and osteoclast-like cells.

Bone is an estradiol-responsive tissue. Estrogen withdrawal during the menopause causes loss of bone mass and clinically relevant osteoporosis in a third of all women. Sufficient or impaired local production, as well as degradation of estradiol in cells present in the bone microenvironment might be an important mechanism of rescue or might contribute to the development of osteoporosis, respectively. We therefore investigated aromatase and 17beta-hydroxysteroid dehydrogenase type IV (17beta-HSD IV) expression in osteoblast- and osteoclast-like cells. Aromatase mRNA was increasingly expressed in myeloid THP 1 cells differentiated along the monocyte/phagocyte pathway exploiting vitamin D and either granulocyte-macrophage-stimulating factor (GMCSF) or macrophage-stimulating factor (MCSF). In long-term cultures, when sequentially exposed to vitamin D (days 0-21) and GMCSF (days 5-10) and plated on collagen, the amount of expression of aromatase mRNA steadily increased along with the increasing expression of osteopontin mRNA, alpha(v) integrin mRNA, c-fms (MCSF-receptor) mRNA and multinucleated cells developing. The conversion of estradiol from testosterone (10(-7) M/l) in the supernatants of dishes mirrored changes in aromatase mRNA expression and by day 21 rose to 30,000 ng/10(7) cells/24 h. 17Beta-HSD IV mRNA expression was abundant in undifferentiated THP 1 cells and was decreased to approximately 50% by day 21. Unstimulated SV-40 immortalized fetal osteoblasts did not express aromatase mRNA, but the expression was stimulated by the addition of the phorbol ester phorbol myristate acetate (PMA). Unstimulated osteoblasts from primary cultures did not express aromatase mRNA. Osteoblast-like osteosarcoma cells MG 63 expressed faint levels of aromatase mRNA in contrast to the osteosarcoma cell line HOS 58. 17Beta-HSD IV mRNA was expressed in fetal osteoblasts as well as in osteoblasts from primary culture, MG 63 and HOS 58 cells. In summary, we can show the expression of estradiol metabolizing enzymes in cells which are present in the bone microenvironment. Impaired aromatase expression and/or enhanced expression of 17beta-HSD IV may contribute to the pathogenesis of osteoporosis.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency.

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.

17-Hydroxysteroid Dehydrogenases↗

PubMed2001

Peroxisome proliferator-activated receptor gamma-mediated transcriptional up-regulation of the hepatocyte growth factor gene promoter via a novel composite cis-acting element.

Hepatocyte growth factor (HGF) is a pleotropic polypeptide that can function as a morphogen, motogen, mitogen, angiogen, carcinogen, and tumor suppressor, depending on the target cell and tissue. Previous studies from our laboratory using transgenic mice have shown that HGF gene expression is tightly regulated at the transcriptional level and that the upstream regulatory elements are crucial for the control of HGF gene transcription. In the present study, we have identified and characterized one of these elements as a peroxisome proliferator-activated receptor gamma (PPARgamma)-responsive element. This regulatory element was localized at -246 to -233 base pairs upstream from the transcription start site of the HGF gene promoter having the sequence GGGCCAGGTGACCT. Gel mobility shift and supershift assays demonstrated that this cis-acting element strongly binds to the PPARgamma isoforms as well as to chicken ovalbumin upstream promoter-transcription factor, a member of the orphan nuclear receptor subfamily. Mutational analysis and gel mobility band shift assays indicated that the binding site is an inverted repeat of the AGGTCA motif with two spacers (inverted repeat 2 configuration) and that the two spacers are important for PPARgamma binding. This binding site overlaps with functional binding sites for activating protein-2, nuclear factor 1, and upstream stimulatory factor, and together, they constitute a multifunctional composite binding site through which these different transcription factors exert their regulatory effects on HGF promoter activity. Functional assays revealed that PPARgamma, with its ligand, 15-deoxy-prostaglandin J2, strongly stimulates HGF promoter activity. On the other hand, nuclear factor 1, activating protein-2, and chicken ovalbumin upstream promoter-transcription factor transcription factors repress the stimulatory action of PPARgamma by competing with PPARgamma for their overlapping binding sites. Furthermore, for the first time, our studies demonstrate that the PPARgamma ligand, 15-deoxy-prostaglandin J2, induces endogenous HGF mRNA and protein expression in fibroblasts in culture.

3T3 Cells↗

PubMed1999

Role and organization of peroxisomal beta-oxidation.

In mammals, peroxisomes are involved in breakdown of very long chain fatty acids, prostanoids, pristanic acid, dicarboxylic fatty acids, certain xenobiotics and bile acid intermediates. Substrate spectrum and specificity studies of the four different beta-oxidation steps in rat and/or in man demonstrate that these substrates are degraded by separate beta-oxidation systems composed of different enzymes. In both species, the enzymes acting on straight chain fatty acids are palmitoyl-CoA oxidase, an L-specific multifunctional protein (MFP-1) and a dimeric thiolase. In liver, bile acid intermediates undergo one cycle of beta-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase (in rat), or branched chain acyl-CoA oxidase (in man), a D-specific multifunctional protein (MFP-2) and SCPX-thiolase. Finally, pristanic acid is degraded in rat tissues by pristanoyl-CoA oxidase, the D-specific multifunctional protein-2 and SCPX-thiolase. Although in man a pristanoyl-CoA oxidase gene is present, so far its product has not been found. Hence, pristanoyl-CoA is believed to be desaturated in human tissues by the branched chain acyl-CoA oxidase. Due to the stereospecificity of the oxidases acting on 2-methyl-branched substrates, an additional enzyme, 2-methylacyl-CoA racemase, is required for the degradation of pristanic acid and the formation of bile acids.

Acyl-CoA Dehydrogenase, Long-Chain↗