Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids, 2001
Because several studies indicated that peroxisomes are important for the biosynthesis of isopreno... more Because several studies indicated that peroxisomes are important for the biosynthesis of isoprenoids, we wanted to investigate whether a reduced availability of isoprenoids could be one of the pathogenic factors contributing to the severe phenotype of the Pex5−/− mouse, a model for Zellweger syndrome. Total cholesterol was determined in plasma, brain and liver of newborn mice. In none of these
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2015
α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-interm... more α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.
Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused rema... more Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp-Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long-chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron-specific peroxisome knockout model, NEX-Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP-Pex5(-/-) mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP-Pex5(-/-) mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning.
To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on fu... more To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on func- tional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for hu- man Zellweger syndrome. We found that all enzymes mea- sured, including putative peroxisomal enzymes, are at least as active in the
The functional diversity and molecular adaptations of reactive microglia in the chronically infla... more The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2(-/-) microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2(-/-) microglia were compared with those from SOD1(G93A) mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1(G93A) spinal cord, neurons were intact in Mfp2(-/-) brain and Mfp2(-/-) microglia lacked signs of phagoc...
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we repor... more The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued ...
Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological fun... more Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological functions. The possible roles of oxysterols in various neurological diseases makes the analysis of these metabolites in the central nervous system of particular interest. Here, we report the identification and quantification of a panel of twelve sterols in mouse cerebrospinal fluid (CSF) using liquid chromatography-mass spectrometry exploiting enzyme assisted derivatisation for sterol analysis technology. We found low levels of oxysterols and cholestenoic acids in CSF in the range of 5pg/mL-2.6ng/mL. As found in man, these concentrations are one to two orders of magnitude lower than in plasma.
Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairm... more Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairment of neuronal migration. Although the molecular mechanisms underlying the neuronal migration defect are at present unknown, the excess of very long chain fatty acids in brain, a consequence of peroxisomal-oxidation deficiency, has often been hypothesized to play a major role. The purpose of the present study was to
The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a ra... more The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.
The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient i... more The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.
The metabolic factors causing cortical neuronal migration defects, hypotonia and malformation of ... more The metabolic factors causing cortical neuronal migration defects, hypotonia and malformation of cerebellum in patients and mice with severe peroxisome biogenesis disorders are still not identified. In the present investigation, we tested the hypothesis that the combined inactivity of peroxisomal beta-oxidation and ether lipid biosynthesis could be at the origin of these pathologies. Double MFP2/DAPAT knockout mice were generated and their postnatal phenotypes were compared with single knockouts and control mice. Cortical neuronal migration was not affected in DAPAT knockouts and only mildly in double MFP2/DAPAT knockout mice. The latter mice were severely hypotonic and usually died in the postnatal period. Both DAPAT and MFP2 single knockout mice exhibited delays in the formation of cerebellar folia. We conclude that the combined defect of peroxisomal beta-oxidation and ether lipid synthesis does not solely account for the typical cortical neuronal migration defect of mice with peroxisome biogenesis disorders but contributes to their hypotonia.
To better delineate the sequence requirements for high affinity binding of retinoid X receptor al... more To better delineate the sequence requirements for high affinity binding of retinoid X receptor alpha (RXR alpha) homodimers, a selection protocol was used starting from a random pool of oligonucleotides. All recovered sequences contained at least two hexamers related to the consensus sequence for the thyroid/retinoid subfamily of nuclear receptors, A/GGGTCA. These hexamers were most frequently organised as direct repeats with one interspacing base pair (DR1) and as palindromic repeats without interspacing base pairs (PAL0), the established configurations for RXR response elements (RXREs). However, DR2 and DR6 configurations also appeared to bind RXR alpha homodimers with high affinity, as did elements consisting of three hexamers. Reporters containing single copies of these elements conferred 9-cis retinoic acid responsiveness to cells cotransfected with an RXR alpha expressing plasmid. The upstream hexamer of all recovered sites was preferentially preceded by a G and its consensus was GGGTCA. Based on the composition of the selected DR1 RXREs, and the functional and mutational analysis, the optimal DR1 RXRE consists of an upstream hexamer starting with A or G and preceded by A or G. The interspacing base can be either G, A or T but not C. The affinity of RXR alpha homodimers for a DR1 element is strongly reduced when the final position is taken by a C. The results of the present investigation indicate that RXR alpha homodimers may have broader DNA binding specificities than currently believed. The biological relevance of these alternative RXREs will need to be corroborated by the identification of natural elements of this kind.
... [3,4-14C]All-rac α-tocopherol (spe-cific activity: 53.75 Ci/mol) was a generous gift from F. ... more ... [3,4-14C]All-rac α-tocopherol (spe-cific activity: 53.75 Ci/mol) was a generous gift from F. Hoff-mann-La ... Hilde Van Houtea, Edmond de Hoffmannb, Paul P. Van Veldhovenc, Guy P. Mannaertsc, Hubert Carchond, Myriam I. Baesa, and Peter E. Declercqa,* aDepartment of ...
According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the... more According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.
Biochimica Et Biophysica Acta-molecular and Cell Biology of Lipids, 2001
Because several studies indicated that peroxisomes are important for the biosynthesis of isopreno... more Because several studies indicated that peroxisomes are important for the biosynthesis of isoprenoids, we wanted to investigate whether a reduced availability of isoprenoids could be one of the pathogenic factors contributing to the severe phenotype of the Pex5−/− mouse, a model for Zellweger syndrome. Total cholesterol was determined in plasma, brain and liver of newborn mice. In none of these
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2015
α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-interm... more α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.
Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused rema... more Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp-Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long-chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron-specific peroxisome knockout model, NEX-Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP-Pex5(-/-) mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP-Pex5(-/-) mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning.
To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on fu... more To unravel the conflicting data concerning the dependence of human cholesterol biosynthesis on func- tional peroxisomes, we determined activities and levels of selected enzymes involved in cholesterol biosynthesis in livers of PEX5 knockout mice, a well-characterized model for hu- man Zellweger syndrome. We found that all enzymes mea- sured, including putative peroxisomal enzymes, are at least as active in the
The functional diversity and molecular adaptations of reactive microglia in the chronically infla... more The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2(-/-) microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2(-/-) microglia were compared with those from SOD1(G93A) mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1(G93A) spinal cord, neurons were intact in Mfp2(-/-) brain and Mfp2(-/-) microglia lacked signs of phagoc...
The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we repor... more The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued ...
Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological fun... more Oxysterols and cholestenoic acids are oxidised forms of cholesterol with a host of biological functions. The possible roles of oxysterols in various neurological diseases makes the analysis of these metabolites in the central nervous system of particular interest. Here, we report the identification and quantification of a panel of twelve sterols in mouse cerebrospinal fluid (CSF) using liquid chromatography-mass spectrometry exploiting enzyme assisted derivatisation for sterol analysis technology. We found low levels of oxysterols and cholestenoic acids in CSF in the range of 5pg/mL-2.6ng/mL. As found in man, these concentrations are one to two orders of magnitude lower than in plasma.
Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairm... more Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairment of neuronal migration. Although the molecular mechanisms underlying the neuronal migration defect are at present unknown, the excess of very long chain fatty acids in brain, a consequence of peroxisomal-oxidation deficiency, has often been hypothesized to play a major role. The purpose of the present study was to
The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a ra... more The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.
The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient i... more The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.
The metabolic factors causing cortical neuronal migration defects, hypotonia and malformation of ... more The metabolic factors causing cortical neuronal migration defects, hypotonia and malformation of cerebellum in patients and mice with severe peroxisome biogenesis disorders are still not identified. In the present investigation, we tested the hypothesis that the combined inactivity of peroxisomal beta-oxidation and ether lipid biosynthesis could be at the origin of these pathologies. Double MFP2/DAPAT knockout mice were generated and their postnatal phenotypes were compared with single knockouts and control mice. Cortical neuronal migration was not affected in DAPAT knockouts and only mildly in double MFP2/DAPAT knockout mice. The latter mice were severely hypotonic and usually died in the postnatal period. Both DAPAT and MFP2 single knockout mice exhibited delays in the formation of cerebellar folia. We conclude that the combined defect of peroxisomal beta-oxidation and ether lipid synthesis does not solely account for the typical cortical neuronal migration defect of mice with peroxisome biogenesis disorders but contributes to their hypotonia.
To better delineate the sequence requirements for high affinity binding of retinoid X receptor al... more To better delineate the sequence requirements for high affinity binding of retinoid X receptor alpha (RXR alpha) homodimers, a selection protocol was used starting from a random pool of oligonucleotides. All recovered sequences contained at least two hexamers related to the consensus sequence for the thyroid/retinoid subfamily of nuclear receptors, A/GGGTCA. These hexamers were most frequently organised as direct repeats with one interspacing base pair (DR1) and as palindromic repeats without interspacing base pairs (PAL0), the established configurations for RXR response elements (RXREs). However, DR2 and DR6 configurations also appeared to bind RXR alpha homodimers with high affinity, as did elements consisting of three hexamers. Reporters containing single copies of these elements conferred 9-cis retinoic acid responsiveness to cells cotransfected with an RXR alpha expressing plasmid. The upstream hexamer of all recovered sites was preferentially preceded by a G and its consensus was GGGTCA. Based on the composition of the selected DR1 RXREs, and the functional and mutational analysis, the optimal DR1 RXRE consists of an upstream hexamer starting with A or G and preceded by A or G. The interspacing base can be either G, A or T but not C. The affinity of RXR alpha homodimers for a DR1 element is strongly reduced when the final position is taken by a C. The results of the present investigation indicate that RXR alpha homodimers may have broader DNA binding specificities than currently believed. The biological relevance of these alternative RXREs will need to be corroborated by the identification of natural elements of this kind.
... [3,4-14C]All-rac α-tocopherol (spe-cific activity: 53.75 Ci/mol) was a generous gift from F. ... more ... [3,4-14C]All-rac α-tocopherol (spe-cific activity: 53.75 Ci/mol) was a generous gift from F. Hoff-mann-La ... Hilde Van Houtea, Edmond de Hoffmannb, Paul P. Van Veldhovenc, Guy P. Mannaertsc, Hubert Carchond, Myriam I. Baesa, and Peter E. Declercqa,* aDepartment of ...
According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the... more According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.
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