Peroxisomal ABC Transporter Research Articles

Pseudo half-molecules of the ABC transporter, COMATOSE, bind Pex19 and target to peroxisomes independently but are both required for activity

Peroxisomal ABC transporters of animals and fungi are “half-size” proteins which dimerise to form a functional transporter. However, peroxisomal ABC transporters of land plants are synthesised as a single polypeptide which represents a fused heterodimer. The N- and C-terminal pseudo-halves of COMATOSE (CTS; AtABCD1) were expressed as separate polypeptides which bound Pex19 in vitro and targeted independently to the peroxisome membrane in yeast, where they were stable but not functional. When co-expressed, the pseudo-halves were fully functional as indicated by ATPase activity and rescue of the pxa1pxa2Δ mutant for growth on oleate. The functional significance of heterodimer asymmetry is discussed. Structured summary of protein interactionsPEX19-1 binds to CTS-N by pull down (View Interaction)PEX19-1 binds to CTS-C by pull down (View Interaction)PEX19-2 binds to CTS-N by pull down (View Interaction)PEX19-2 binds to CTS-N by pull down (View Interaction)

HDAC inhibitor SAHA normalizes the levels of VLCFAs in human skin fibroblasts from X-ALD patients and downregulates the expression of proinflammatory cytokines in Abcd1/2-silenced mouse astrocytes

X-adrenoleukodystrophy (X-ALD) is a peroxisomal metabolic disorder caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). The consistent metabolic abnormality in all forms of X-ALD is an inherited defect in the peroxisomal β-oxidation of very long chain FAs (VLCFAs >C22:0) and the resultant pathognomic accumulation of VLCFA. The accumulation of VLCFA leads to a neuroinflammatory disease process associated with demyelination of the cerebral white matter. The present study underlines the importance of a potent histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA) in inducing the expression of ABCD2 [adrenoleukodystrophy-related protein (ALDRP)], and normalizing the peroxisomal β-oxidation, as well as the saturated and monounsaturated VLCFAs in cultured human skin fibroblasts of X-ALD patients. The expression of ELOVL1, the single elongase catalyzing the synthesis of both saturated VLCFA (C26:0) and monounsaturated VLCFA (C26:1), was also reduced by SAHA treatment. In addition, using Abcd1/Abcd2-silenced mouse primary astrocytes, we also examined the effects of SAHA in VLCFA-induced inflammatory response. SAHA treatment decreased the inflammatory response as expression of inducible nitric oxide synthase, inflammatory cytokine, and activation of NF-κB in Abcd1/Abcd2-silenced mouse primary astrocytes was reduced. These observations indicate that SAHA corrects both the metabolic disease of VLCFA as well as secondary inflammatory disease; therefore, it may be an ideal drug candidate to be tested for X-ALD therapy in humans.

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.

Peroxisomal transporters associated with β-oxidation

Peroxisomes perform a range of different functions, including β-oxidation of fatty acids and synthesis and degradation of bioactive lipid-derived molecules. A key feature of peroxisomes is their role in metabolic pathways which are shared between several subcellular compartments, including mitochondria, chloroplasts and cytosol. Transport across the peroxisomal membrane is therefore essential for the coordination of metabolism. Although transport proteins are very likely required for import of substrates and cofactors, export of intermediates and products and the operation of redox shuttles, relatively few peroxisomal transporters have been identified to date. We have identified and characterised two peroxisomal transport systems which are required for β-oxidation in the model plant, Arabidopsis thaliana. Peroxisomal Nucleotide Carrier 1 and 2 were identified by homology with the yeast peroxisomal adenine nucleotide carrier and were shown by complementation and in vitro uptake assays to catalyse the counter exchange of ATP with AMP [1]. Inducible RNAi lines demonstrated that import of ATP into peroxisomes is essential for activation of fatty acids during seedling establishment and plays a role in other β-oxidation reactions such as auxin metabolism. Arabidopsis also contains a single peroxisomal ABC transporter, COMATOSE (CTS), which has been identified in at least four independent forward genetic screens. Analysis of cts null mutants has demonstrated that CTS plays key roles in a number of developmental and physiological processes, including germination, seedling establishment, fertility and root growth [2]. We demonstrate that the different roles of CTS in planta are separable by mutagenesis [3] and can be related to different biochemical roles, specifically the ability to metabolise distinct substrates such as fatty acids and hormone precursors via β-oxidation. Taken together, these findings strongly suggest that CTS is a broad specificity transporter which mediates uptake of substrates for β-oxidation into the peroxisome. Here, we present biochemical characterisation of heterologously-expressed CTS, provide evidence for its role as a transporter of fatty acyl-CoAs and compare its activity to that of yeast and mammalian homologues.

A peroxisomal ABC transporter promotes seed germination by inducing pectin degradation under the control of ABI5

Seed dormancy is essential for most plants to control the timing of germination. In Arabidopsis thaliana, PED3 is a single-copy gene encoding an ATP-binding cassette transporter that is required for peroxisomal fatty acid beta-oxidation. PED3 is involved in the import of several biologically important molecules into the peroxisome, including very-long-chain fatty acids associated with the breakdown of seed-reserve lipids, and precursors of auxin and jasmonic acid. The germination of ped3 mutants is significantly impaired, suggesting that PED3 regulates dormancy and germination. A transcriptome analysis revealed that many genes containing the core motif of the ABA responsive element (ABRE) in their promoter regions, and the ABA insensitive 5 (ABI5) transcription factor that binds to ABRE, are abnormally up-regulated in imbibed ped3 seeds. Expression of polygalacturonase inhibiting proteins (PGIPs) is also up-regulated specifically in ped3 after imbibition. By contrast, the ped3 abi5 double mutant does not show any of these expression patterns. The results indicate that the abi5 mutation normalizes PGIP expression and rescues the impaired germination phenotype of the ped3 mutant. PGIPs are known to act as inhibitors of polygalacturonases that degrade pectin. The amount of PGIP1 transcript regulates the timing of radicle protrusion. The impaired germination of ped3 could also be rescued by removal of pectin from the seed coat using exogenous polygalacturonase or acidic conditions. Overall, our results suggest that PED3, a peroxisomal ABC transporter, promotes seed germination by suppressing PGIPs under the control of ABI5.

The role of ELOVL1 in very long‐chain fatty acid homeostasis and X‐linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). X-ALD is characterized by the accumulation of very long-chain fatty acids (VLCFA; ≥C24) in plasma and tissues. In this manuscript we provide insight into the pathway underlying the elevated levels of C26:0 in X-ALD. ALDP transports VLCFacyl-CoA across the peroxisomal membrane. A deficiency in ALDP impairs peroxisomal β-oxidation of VLCFA but also raises cytosolic levels of VLCFacyl-CoA which are substrate for further elongation. We identify ELOVL1 (elongation of very-long-chain-fatty acids) as the single elongase catalysing the synthesis of both saturated VLCFA (C26:0) and mono-unsaturated VLCFA (C26:1). ELOVL1 expression is not increased in X-ALD fibroblasts suggesting that increased levels of C26:0 result from increased substrate availability due to the primary deficiency in ALDP. Importantly, ELOVL1 knockdown reduces elongation of C22:0 to C26:0 and lowers C26:0 levels in X-ALD fibroblasts. Given the likely pathogenic effects of high C26:0 levels, our findings highlight the potential of modulating ELOVL1 activity in the treatment of X-ALD.

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves ofArabidopsisduring Extended Darkness

Fatty acid beta-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core beta-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal beta-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly alpha-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable alpha-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of alpha-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and beta-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal beta-oxidation plays a major role in dark-treated plants after depletion of starch reserves.

Mutations in theArabidopsisPeroxisomal ABC Transporter COMATOSE Allow Differentiation between Multiple Functions In Planta: Insights from an Allelic Series

COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal beta-oxidation. A new allelic series and a homology model based on the bacterial ABC transporter, Sav1866, provide novel insights into structure-function relations of ABC subfamily D proteins. In contrast to ALDP, where the majority of mutations result in protein absence from the peroxisomal membrane, all CTS mutants produced stable protein. Mutation of conserved residues in the Walker A and B motifs in CTS nucleotide-binding domain (NBD) 1 resulted in a null phenotype but had little effect in NBD2, indicating that the NBDs are functionally distinct in vivo. Two alleles containing mutations in NBD1 outside the Walker motifs (E617K and C631Y) exhibited resistance to auxin precursors 2,4-dichlorophenoxybutyric acid (2,4-DB) and indole butyric acid (IBA) but were wild type in all other tests. The homology model predicted that the transmission interfaces are domain-swapped in CTS, and the differential effects of mutations in the conserved "EAA motif" of coupling helix 2 supported this prediction, consistent with distinct roles for each NBD. Our findings demonstrate that CTS functions can be separated by mutagenesis and the structural model provides a framework for interpretation of phenotypic data.

Peroxisomal ABC transporters and β-oxidation during the life cycle of the filamentous fungus Podospora anserina

ATP-binding cassette transporters are ubiquitous proteins that facilitate transport of diverse substances across a membrane. However, their exact role remains poorly understood. In order to test their function in a fungus life cycle, we deleted the two Podospora anserina peroxisomal ABC transporter pABC1 and pABC2 genes as well as the three genes involved in peroxisomal ( fox2) and mitochondrial ( scdA and echA) β-oxidation. Analysis of the single and double mutants shows that fatty acid β-oxidation occurs in both organelles. Furthermore, the peroxisomal and mitochondrial fatty acid β-oxidation pathways are both dispensable for vegetative and sexual development. They are, however, differently required for ascospore pigmentation and germination, this latter defect being restored in a ΔpABC1 and ΔpABC2 background . We report also that lack of peroxisomal ABC transporters does not prevent peroxisomal long-chain fatty acid oxidation, suggesting the existence of another pathway for their import into peroxisomes. Finally, we show that some aspects of fatty acid degradation are clearly fungus species specific.

Distinct modulatory roles for thyroid hormone receptors TR α and TR β in SREBP1-activated ABCD2 expression

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone ( T 3) receptor (TR) α and TR β bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TR β, but not TR α, represses ABCD2 induction independently of DNA binding. However, activation by TR α and derepression of TR β are T 3-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T 3 and TR α levels) but downregulated in adults (with low T 3 and TR α but elevated TR β levels). This temporal repression of Abcd2 is blunted in TR β-deficient mice, and the response to manipulated T 3 states is abrogated in TR α-deficient mice. These findings show that TR α and TR β differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.

Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters

Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (α-linolenic acid, 18:3 n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6 n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 ( Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism.

Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Dehydroepiandrosterone up-regulates the Adrenoleukodystrophy-related gene ( ABCD2) independently of PPARα in rodents

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC transporter, ALDP, supposed to participate in the transport of very long chain fatty acids (VLCFA). The adrenoleukodystrophy-related protein (ALDRP), which is encoded by the ABCD2 gene, is the closest homolog of ALDP and is considered as a potential therapeutic target since functional redundancy has been demonstrated between the two proteins. Pharmacological induction of Abcd2 by fibrates through the activation of PPARα has been demonstrated in rodent liver. DHEA, the most abundant steroid in human, is described as a PPARα activator and also as a prohormone able to mediate induction of several genes. Here, we explored the in vitro and in vivo effects of DHEA on the expression of peroxisomal ABC transporters. We show that Abcd2 and Abcd3 but not Abcd4 are induced in primary culture of rat hepatocytes by DHEA-S. We also demonstrate that Abcd2 and Abcd3 but not Abcd4 are inducible by an 11-day treatment with DHEA in the liver of male rodents but not in brain, testes and adrenals. Finally and contrary to Abcd3, we show that the mechanism of induction of Abcd2 is independent of PPARα.

The role of peroxisomal ABC transporters in the mouse adrenal gland: the loss of Abcd2 (ALDR), Not Abcd1 (ALD), causes oxidative damage

X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that primarily affects the adrenal cortex, as well as myelin and axons of the central nervous system. Marked phenotypic heterogeneity does not correlate with disease-causing mutations in ABCD1, which encodes a peroxisomal membrane protein that is a member of the ABC transmembrane transporter proteins. The precise physiological functions of ABCD1 and ABCD2, a closely related peroxisomal membrane half-transporter, are unknown. The abcd1 knockout mouse does not develop the inflammatory demyelination so typical and devastating in adreno-leukodystrophy, but it does display the same lamellae and lipid profiles in adrenocortical cells under the electron microscope as the human patients. The adrenocortical cells in the mouse also exhibit immunohistochemical evidence of oxidative stress at 12 weeks but no evidence of oxidative damage. To better understand the pathogenesis of this complex disease, we evaluate the adrenal lesion of the abcd1 knockout mouse as a function of normal aging, dietary or therapeutic manipulations, and abcd genotype. The loss of abcd2 causes oxidative stress in the adrenal at 12 weeks, as judged by increased immunoreactivity for the mitochondrial manganese superoxide dismutase, in both the inner cortex and medulla. The loss of abcd2 (n=20), but not abcd1 (n=27), results in the spontaneous and premature deposition of ceroid, a known end-product of oxidative damage, predominantly in adrenal medullary cells. These data indicate that the loss of abcd2 results in greater oxidative stress in murine adrenal cells than the loss of abcd1, providing a clue to its cellular function. We also find that the adrenocortical lesion of the abcd1 knockout mouse does not produce functional impairment at ten to nineteen months or overt hypocortisolism at any age, nor does it progress histologically; these and other data align this mouse model closer to human female heterozygotes than to male ALD or AMN hemizygotes.

AXONAL ABNORMALITIES IN THE BRAIN OF THE ABCD1 KNOCKOUT MOUSE, AN ANIMAL MODEL OF X-LINKED ADRENOLEUKODYSTROPHY

Objectives: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding a peroxisomal ABC transporter. Mice that carry the ABCD1 gene defect develop a late onset phenotype that appears comparable to adrenomyeloneuropathy (AMN), the adult form of X-ALD. We set out to examine the brain of the ABCD1 knockout mouse by diffusion tensor imaging (DTI) and ultrastructural analysis.

Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism

The Arabidopsis acyl-CoA oxidase (ACX) family comprises isozymes with distinct fatty acid chain-length specificities that together catalyse the first step of peroxisomal fatty acid beta-oxidation. We have isolated and characterized T-DNA insertion mutants in the medium to long-chain (ACX1) and long-chain (ACX2) acyl-CoA oxidases, and show that the corresponding endogenous activities are decreased in the mutants. Lipid catabolism during germination and early post-germinative growth was unaltered in the acx1-1 mutant, but slightly delayed in the acx2-1 mutant, with 3-day-old acx2-1 seedlings accumulating long-chain acyl-CoAs. In acx1-1 and acx2-1, seedling growth and establishment in the absence of an exogenous supply of sucrose was unaffected. Seedlings of the double mutant acx1-1 acx2-1 were unable to catabolize seed storage lipid, and accumulated long-chain acyl-CoAs. The acx1-1 acx2-1 seedlings were also unable to establish photosynthetic competency in the absence of an exogenous carbon supply, a phenotype that is shared with a number of other Arabidopsis mutants disrupted in storage lipid breakdown. Germination frequency of the double mutant was significantly reduced compared with wild-type seeds. This was unaffected by the addition of exogenous sucrose, but was improved by dormancy-breaking treatments such as cold stratification and after-ripening. We show that the acx1-1, acx2-1 and acx1-2 acx2-1 double mutants and the ketoacyl-CoA thiolase-2 (kat2) mutant exhibit a sucrose-independent germination phenotype comparable with that reported for comatose (cts-2), a mutant in a peroxisomal ABC transporter which exhibits enhanced dormancy. This demonstrates an additional role beyond that of carbon provision for the beta-oxidation pathway during germination or in dormant seeds.

Targeting of the human adrenoleukodystrophy protein to the peroxisomal membrane by an internal region containing a highly conserved motif

In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein. This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood. Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting. However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself. Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency. Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome. The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1. Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting. The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency. The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria. Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.

Thyroid hormone induction of the adrenoleukodystrophy-related gene (ABCD2).

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disorder associated with impaired very-long-chain fatty-acid (VLCFA) beta-oxidation caused by mutations in the ABCD1 (ALD) gene that encodes a peroxisomal membrane ABC transporter. ABCD2 (ALDR) displays partial functional redundancy because when overexpressed, it is able to correct the X-ALD biochemical phenotype. The ABCD2 promoter contains a putative thyroid hormone-response element conserved in rodents and humans. In this report, we demonstrate that the element is capable of binding retinoid X receptor and 3,5,3'-tri-iodothyronine (T3) receptor (TRbeta) as a heterodimer and mediating T3 responsiveness of ABCD2 in its promoter context. After a T3 treatment, an induction of the ABCD2 gene was observed in the liver of normal rats but not that of TRbeta-/- mice. ABCD2 was not induced in the brain of the T3-treated rats. However, we report for the first time that induction of the ABCD2 redundant gene is feasible in myelin-producing cells (differentiated CG4 oligodendrocytes). The induction was specific for this cell type because it did not occur in astrocytes. Furthermore, we observed T3 induction of ABCD2 in human and mouse ABCD1-deficient fibroblasts, which was correlated with normalization of the VLCFA beta-oxidation. Finally, ABCD3 (PMP70), a close homolog of ABCD2, was also induced by T3 in the liver of control rats, but not that of TRbeta-/- mice, and in CG4 oligodendrocytes.

Evidence against the adrenoleukodystrophy-related gene acting as a modifier of X-adrenoleukodystrophy.

Mutations in the ALD gene encoding a peroxisomal ABC-transporter are the cause of X-linked adrenoleukodystrophy (X-ALD). The clinical course of the disease is extremely heterogeneous, ranging from severe phenotypes leading to death in early childhood to asymptomatic forms. Mutation analysis in X-ALD patients showed no predictable genotype-phenotype correlation. Out of 3 peroxisomal ABC-transporters the human adrenoleukodystrophy related protein (hALDRP, Holzinger et al. 1997) displays the highest homology to the adrenoleukodystrophy protein (ALDP). ALDRP can restore P-oxidation in X-ALD fibroblasts in in vitro expression experiments and directly interacts with ALDP.KeywordsSegregation AnalysisTranslational Start CodonPutative Promoter RegionChildhood FormFrequent PolymorphismThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Dehydroepiandrosterone induction of the Abcd2 and Abcd3 genes encoding peroxisomal ABC transporters: implications for X-linked adrenoleukodystrophy.

Dehydroepiandrosterone (DHEA) is a peroxisome proliferator known to increase the expression of the genes encoding the peroxisomal ß-oxidation enzymes in rodents. Using RT-PCR, we analysed the expression of the Abcd2 and Abcd3 genes encoding the peroxisomal ABC transporters ALDRP (ALD related protein) and PMP70 (70 kDa peroxisomal membrane protein) in primary cultures of rats hepatocytes treated with sulfated DHEA. We observed a time (12-72h) and dose (125-500μM) dependent increase in the expression of both genes.KeywordsAdrenal InsufficiencyPeroxisome ProliferatorIndirect PathwayDependent IncreaseAbed3 GeneThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.