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Peroxisomes play a crucial role in normal neurological development as exemplified by the devastating neurological consequences of a defect in the biogenesis of peroxisomes as in Zellweger syndrome. The underlying basis for the important role of peroxisomes in neurological development resides in the fact that peroxisomes catalyze a number of physiological functions, notably involving the metabolism of different lipids. Indeed, peroxisomes catalyse the beta-oxidative breakdown of certain fatty acids including: (1.) the very long-chain fatty acids C22:0, C24:0, and C26:0; (2.) pristanic acid and (3.) the bile acid intermediates di- and trihydroxycholestanoic acid which cannot be oxidized in mitochondria. Furthermore, peroxisomes catalyze the synthesis of a particular type of lipids, i.e. ether-linked phospholipids, which are highly abundant in brain, especially in myelin. The current state of knowledge with respect to the metabolic role of peroxisomes will be described in this paper with particular emphasis on the role of peroxisomes in brain., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Peroxisomes are dynamic organelles that play an essential role in a variety of cellular catabolic and anabolic metabolic pathways, including fatty acid alpha- and beta-oxidation, and plasmalogen and bile acid synthesis. Defects in genes encoding peroxisomal proteins can result in a large variety of peroxisomal disorders either affecting specific metabolic pathways, i.e., the single peroxisomal enzyme deficiencies, or causing a generalized defect in function and assembly of peroxisomes, i.e., peroxisome biogenesis disorders. In this review, we discuss the clinical, biochemical, and genetic aspects of all human peroxisomal disorders currently known., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Import of peroxisomal matrix proteins, crucial for peroxisome biogenesis, is mediated by the cytosolic receptors PEX5 and PEX7 that recognize proteins carrying peroxisomal targeting signals 1 or 2 (PTS1 or PTS2), respectively. Mutations in PEX5 or 12 other PEX genes cause peroxisome biogenesis disorders, collectively named the Zellweger spectrum disorders (ZSDs), whereas mutations in PEX7 cause rhizomelic chondrodysplasia punctata type 1 (RCDP1). Three additional RCDP types, RCDP2-3-4, are caused, respectively, by mutations in GNPAT, AGPS and FAR1, encoding enzymes involved in plasmalogen biosynthesis. Here we report a fifth type of RCDP (RCDP5) caused by a novel mutation in PEX5. In four patients with RCDP from two independent families, we identified a homozygous frame shift mutation c.722dupA (p.Val242Glyfs(∗)33) in PEX5 (GenBank: NM_001131023.1). PEX5 encodes two isoforms, PEX5L and PEX5S, and we show that the c.722dupA mutation, located in the PEX5L-specific exon 9, results in loss of PEX5L only. Both PEX5 isoforms recognize PTS1-tagged proteins, but PEX5L is also a co-receptor for PTS2-tagged proteins. Previous patients with PEX5 mutations had ZSD, mainly due to deficient import of PTS1-tagged proteins. Similarly to mutations in PEX7, loss of PEX5L results in deficient import of PTS2-tagged proteins only, thus causing RCDP instead of ZSD. We demonstrate that PEX5L expression restores the import of PTS2-tagged proteins in patient fibroblasts. Due to the biochemical overlap between RCDP1 and RCDP5, sequencing of PEX7 and exon 9 in PEX5 should be performed in patients with a selective defect in the import of PTS2-tagged proteins., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peroxisomes are subcellular organelles which are present in virtually every eukaryotic cell and catalyze a large number of metabolic functions. The importance of peroxisomes for humans is stressed by the existence of a large group of genetic diseases in which either the biogenesis of peroxisomes is impaired or one of its metabolic functions. Thanks to the work on Zellweger syndrome which is the prototype of the group of peroxisomal disorders, much has been learned about the metabolism and biogenesis of peroxisomes in humans. These metabolic functions include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis; (3.) fatty acid alpha-oxidation, and (4.) glyoxylate detoxification. Since peroxisomes lack a citric acid cycle and a respiratory chain, peroxisomes are relatively helpless organelles which rely heavily on their cross-talk with other subcellular organelles in order to metabolize the end products of metabolism as generated in peroxisomes. The metabolic functions of peroxisomes in humans will be briefly described in this review with emphasis on the cross-talk with other subcellular organelles as well as the peroxisomal disorders in which one or more peroxisomal functions are impaired., (Copyright © 2013. Published by Elsevier Masson SAS.)

We studied the chronological lifespan of glucose-grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome-deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β-oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β-oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β-oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β-oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Background: Zellweger spectrum disorders (ZSDs) are multisystem genetic disorders caused by a lack of functional peroxisomes, due to mutations in one of the PEX genes, encoding proteins involved in peroxisome biogenesis. The phenotypic spectrum of ZSDs ranges from an early lethal form to much milder presentations. In cultured skin fibroblasts from mildly affected patients, peroxisome biogenesis can be partially impaired which results in a mosaic catalase immunofluorescence pattern. This peroxisomal mosaicism has been described for specific missense mutations in various PEX genes. In cell lines displaying peroxisomal mosaicism, peroxisome biogenesis can be improved when these are cultured at 30°C. This suggests that these missense mutations affect the folding and/or stability of the encoded protein. We have studied if the function of mutant PEX1, PEX6 and PEX12 can be improved by promoting protein folding using the chemical chaperone arginine., Methods: Fibroblasts from three PEX1 patients, one PEX6 and one PEX12 patient were cultured in the presence of different concentrations of arginine. To determine the effect on peroxisome biogenesis we studied the following parameters: number of peroxisome-positive cells, levels of PEX1 protein and processed thiolase, and the capacity to β-oxidize very long chain fatty acids and pristanic acid., Results: Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX1, 6 and 12 can be improved by arginine., Conclusion: Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisome biogenesis disorder.

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload., (© 2013.)

Peroxisomes play a key role in human physiology as exemplified by the devastating consequences of a defect in peroxisome biogenesis as observed in patients affected by Zellweger syndrome. The main metabolic functions of peroxisomes in humans include: (1) fatty acid beta-oxidation; (2) etherphospholipid synthesis; (3) bile acid synthesis; (4) fatty acid alpha-oxidation, and (5) glyoxylate detoxification. Since peroxisomes lack a citric acid cycle and respiratory chain like mitochondria do, metabolism in peroxisomes requires continued cross-talk with other organelles, notably mitochondria and the endoplasmic reticulum in order to allow continued metabolism of the products generated by peroxisomes. Many of the metabolites which require peroxisomes for homeostasis, are involved in signal transduction pathways. These include the primary bile acids; platelet activating factor; plasmalogens, N-acylglycines and N-acyltaurines; docosahexaenoic acid as well as multiple prostanoids. The current state of knowledge in this area will be discussed in this review.

The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+) becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

The group of peroxisomal disorders represents a growing number of genetically determined diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided in two major subgroups including (1) the peroxisome biogenesis disorders (PBDs) and (2) the single peroxisomal enzyme deficiencies. Liver pathology is a frequent finding in patients affected by a peroxisomal disorder. This is not only true for patients affected by a PBD, but also for patients with a single enzyme defect in one of the metabolic pathways in which peroxisomes are involved. By comparing the different peroxisomal disorders, we provide evidence suggesting that the main hepatotoxic metabolites responsible for the liver pathology found in patients, are the bile acid synthesis intermediates di- and trihydroxycholestanoic acid (DHCA and THCA). Studies in different experimental systems have shown that DHCA and THCA, especially in the unconjugated form, interfere with different physiological processes including mitochondrial oxidative phosphorylation. The implications of these findings will be discussed with special emphasis on patients with di- and trihydroxycholestanoic acidaemia.

Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy, which is caused by mutations in ABCD1. The biochemical hallmark of X-linked adrenoleukodystrophy is the accumulation of very long chain fatty acids (VLCFAs) due to impaired peroxisomal β-oxidation. Although this suggests a role of ABCD1 in VLCFA import into peroxisomes, no direct experimental evidence is available to substantiate this. To unravel the mechanism of peroxisomal VLCFA transport, we use Saccharomyces cerevisiae as a model organism. Here we provide evidence that in this organism very long chain acyl-CoA esters are hydrolyzed by the Pxa1p-Pxa2p complex prior to the actual transport of their fatty acid moiety into the peroxisomes with the CoA presumably being released into the cytoplasm. The Pxa1p-Pxa2p complex functionally interacts with the acyl-CoA synthetases Faa2p and/or Fat1p on the inner surface of the peroxisomal membrane for subsequent re-esterification of the VLCFAs. Importantly, the Pxa1p-Pxa2p complex shares this molecular mechanism with HsABCD1 and HsABCD2.

Peroxisomes are indispensable organelles in higher eukaryotes. They are essential for a number of important metabolic pathways, including fatty acid α- and β-oxidation, and biosynthesis of etherphospholipids and bile acids. However, the peroxisomal membrane forms an impermeable barrier to these metabolites. Therefore, peroxisomes need specific transporter proteins to transfer these metabolites across their membranes. The mammalian peroxisomal membrane harbours three ATP-binding cassette (ABC) transporters. In recent years, significant progress has been made in unravelling the functions of these ABC transporters. There is ample evidence that they are involved in the transport of very long-chain fatty acids, pristanic acid, di- and trihydroxycholestanoic acid, dicarboxylic acids and tetracosahexaenoic acid (C24:6ω3). Surprisingly, only one disease is associated with a deficiency of a peroxisomal ABC transporter. Mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein are the cause for X-linked adrenoleukodystrophy, an inherited metabolic storage disorder. This review describes the current state of knowledge on the mammalian peroxisomal ABC transporters with a particular focus on their function in metabolite transport., (© 2011 Academic Medical Center & Rothamsted Research Ltd. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some 'protein-absent' mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid β-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.

The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Zellweger syndrome spectrum disorders are caused by mutations in any of at least 12 different PEX genes. This includes PEX16, which encodes an integral peroxisomal membrane protein involved in peroxisomal membrane assembly. PEX16-defective patients have been reported to have a severe clinical presentation. Fibroblasts from these patients displayed a defect in the import of peroxisomal matrix and membrane proteins, resulting in a total absence of peroxisomal remnants., Objective: To report on six patients with an unexpected mild variant peroxisome biogenesis disorder due to mutations in the PEX16 gene. Patients presented in the preschool years with progressive spastic paraparesis and ataxia (with a characteristic pattern of progressive leucodystrophy and brain atrophy on MRI scan) and later developed cataracts and peripheral neuropathy. Surprisingly, their fibroblasts showed enlarged, import-competent peroxisomes., Results: Plasma analysis revealed biochemical abnormalities suggesting a peroxisomal disorder. Biochemical variables in fibroblasts were only mildly abnormal or within the normal range. Immunofluorescence microscopy revealed the presence of import-competent peroxisomes, which were increased in size but reduced in number. Subsequent sequencing of all known PEX genes revealed five novel apparent homozygous mutations in the PEX16 gene., Conclusions: An unusual variant peroxisome biogenesis disorder caused by mutations in the PEX16 gene, with a relatively mild clinical phenotype and an unexpected phenotype in fibroblasts, was identified. Although PEX16 is involved in peroxisomal membrane assembly, PEX16 defects can present with enlarged import-competent peroxisomes in fibroblasts. This is important for future diagnostics of patients with a peroxisomal disorder.

Peroxisomes play an essential role in cellular lipid metabolism as exemplified by the existence of a number of genetic diseases in humans caused by the impaired function of one of the peroxisomal enzymes involved in lipid metabolism. Key pathways in which peroxisomes are involved include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis, and (3.) fatty acid alpha-oxidation. In this paper we will describe these different pathways in some detail and will provide an overview of peroxisomal disorders of metabolism and in addition discuss the toxicity of the intermediates of peroxisomal metabolism as they accumulate in the different peroxisomal deficiencies., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The peroxisomal 3-ketoacyl-CoA thiolase B (Thb) gene was previously identified as a direct target gene of PPARalpha, a nuclear hormone receptor activated by hypolipidemic fibrate drugs. To better understand the role of ThB in hepatic lipid metabolism in mice, Sv129 wild-type and Thb null mice were fed or not the selective PPARalpha agonist Wy14,643 (Wy). Here, it is shown that in contrast to some other mouse models deficient for peroxisomal enzymes, the hepatic PPARalpha signaling cascade in Thb null mice was normal under regular conditions. It is of interest that the hypotriglyceridemic action of Wy was reduced in Thb null mice underlining the conclusion that neither thiolase A nor SCPx/SCP2 thiolase can fully substitute for ThB in vivo. Moreover, a significant increased in the expression of lipogenic genes such as Stearoyl CoA Desaturase-1 (SCD1) was observed in Thb null mice fed Wy. Elevation of Scd1 mRNA and protein levels led to higher SCD1 activity, through a molecular mechanism that is probably SREBP1 independent. In agreement with higher SCD1, enrichment of liver mono-unsaturated fatty acids of the n-7 and n-9 series was found in Thb null mice fed Wy. Overall, we show that the reduced peroxisomal beta-oxidation of fat observed in Thb null mice fed Wy is associated with enhanced hepatic lipogenesis, through the combined elevation of microsomal SCD1 protein and activity. Ultimately, not only the amount but also the quality of the hepatic fatty acid pool is modulated upon the deletion of Thb.

It is well established that peroxisomes play a crucial role in de novo bile acid synthesis. Studies in patients with a peroxisomal disorder have been indispensable for the elucidation of the precise role of peroxisomes. Several peroxisomal disorders are associated with distinct bile acid abnormalities and each disorder has a characteristic pattern of abnormal bile acids that accumulate, which is often used for diagnostic purposes. The patients have also been important for determining the pathophysiological consequences of defects in bile acid biosynthesis. In this review, we will discuss all the peroxisomal steps involved in bile acid synthesis and the bile acid abnormalities in patients with peroxisomal disorders. We will show the results of bile acid measurements in several tissues from patients, including brain, and we will discuss the toxicity and the pathological effects of the abnormal bile acids.

Peroxisomes play an important role in bile acid biosynthesis because the last steps of the synthesis pathway are performed by the beta-oxidation system located inside peroxisomes. As a consequence, C(27)-bile acid intermediates accumulate in several peroxisomal disorders. It has been suggested that C(27)-bile acids are especially toxic and contribute to the liver disease associated with peroxisomal disorders. For this reason, we investigated the toxicity of C(27)-bile acids and the underlying mechanisms. We studied the effects of conjugated and unconjugated C(27)-bile acids on cell viability, mitochondrial respiratory chain function and production of oxygen radicals in the rat hepatoma cell line McA-RH7777. Cell viability decreased progressively after incubation with increasing concentrations of different bile acids with dihydroxycholestanoic acid (DHCA) being clearly the most cytotoxic bile acid. In addition, the different bile acids caused a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate and glutamate. Finally, there was a dose-dependent stimulation of ROS generation in the presence of C(27)-bile acids. In conclusion, our studies showed that C(27)-bile acids are more cytotoxic than mature C(24)-bile acids. In addition, C(27)-bile acids are potent inhibitors of oxidative phosphorylation and enhance mitochondrial ROS production by inhibiting the respiratory chain.

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcd1 peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and omega9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA beta-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders.

Proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies on a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder (PBD) identified 11 cell lines with a defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid (VLCFA) concentrations, peroxisomal beta-and alpha-oxidation, dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity, peroxisomal thiolase, and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed 11 different mutations, eight of which are novel. PTS1- and PTS2-protein import capacity was assessed by transfection of the cells with green fluorescent protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines., (Copyright 2008 Wiley-Liss, Inc.)

Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.

Motivation: One important area of clinical genomics research involves the elucidation of molecular mechanisms underlying (complex) disorders which eventually may lead to new diagnostic or drug targets. To further advance this area of clinical genomics one of the main challenges is the acquisition and integration of data, information and expert knowledge for specific biomedical domains and diseases. Currently the required information is not very well organized but scattered over biological and biomedical databases, basic text books, scientific literature and experts' minds and may be highly specific, heterogeneous, complex and voluminous., Results: We present a new framework to construct knowledge bases with concept maps for presentation of information and the web ontology language OWL for the representation of information. We demonstrate this framework through the construction of a peroxisomal knowledge base, which focuses on four key peroxisomal pathways and several related genetic disorders. All 155 concept maps in our knowledge base are linked to at least one other concept map, which allows the visualization of one big network of related pieces of information., Availability: The peroxisome knowledge base is available from www.bioinformaticslaboratory.nl (Support-->Web applications)., Supplementary Information: Supplementary data is available from www.bioinformaticslaboratory.nl (Research-->Output--> Publications--> KB_SuppInfo)

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.

In the present paper, we describe the current state of knowledge regarding the enzymology of the phytanic acid alpha-oxidation pathway. The product of phytanic acid alpha-oxidation, i.e. pristanic acid, undergoes three cycles of beta-oxidation in peroxisomes after which the products, including 4,8-dimethylnonanoyl-CoA, propionyl-CoA and acetyl-CoA, are exported from the peroxisome via one of two routes, including (i) the carnitine-dependent route, mediated by CRAT (carnitine acetyltransferase) and CROT (carnitine O-octanoyltransferase), and (ii) the free acid route, mediated by one or more of the peroxisomal ACOTs (acyl-CoA thioesterases). We also describe our recent data on the omega-oxidation of phytanic acid, especially since pharmacological up-regulation of this pathway may form the basis of a new treatment strategy for ARD (adult Refsum's disease). In patients suffering from ARD, phytanic acid accumulates in tissues and body fluids due to a defect in the alpha-oxidation system.

It is well established that peroxisomes play a crucial role in de novo bile acid biosynthesis. The primary bile acids resulting from peroxisomal beta-oxidation are conjugated to either glycine or taurine in the peroxisomal lumen by a bile acid aminotransferase (BAT). These conjugated bile acids are subsequently secreted into the bile. In this paper we show that the export of glycine- and taurine-conjugated bile acids from mammalian peroxisomes proceeds via specific transporter. The transport activity of this protein was detected by reconstitution of peroxisomal membrane proteins in liposomes and measuring the uptake of radiolabeled substrates into these proteoliposomes. The transporter was further characterized using this assay, which led to the identification of DIDS as an inhibitor of the peroxisomal bile-acid transporter, and allowed us to establish some kinetic parameters for the transport activity.

We report on a newborn girl with microcephaly, abnormal brain development, optic atrophy and hypoplasia, persistent lactic acidemia, and a mildly elevated plasma concentration of very-long-chain fatty acids. We found a defect of the fission of both mitochondria and peroxisomes, as well as a heterozygous, dominant-negative mutation in the dynamin-like protein 1 gene (DLP1). The DLP1 protein has previously been implicated, in vitro, in the fission of both these organelles. Overexpression of the mutant DLP1 in control cells reproduced the fission defect. Our findings are representative of a class of disease characterized by defects in both mitochondria and peroxisomes., (Copyright 2007 Massachusetts Medical Society.)

This review describes the current state of knowledge about the ABCD family of peroxisomal half adenosine-triphosphate-binding cassette (ABC) transporters. ABCDs are predicted to be present in a variety of eukaryotic organisms, although at present, only ABCDs in the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and different mammalian species have been identified and characterized to any significant extent. The functional role of none of these ABCDs has been established definitively and awaits successful reconstitution of ABCDs, either as homo- or heterodimers into liposomes, followed by transport studies. Data obtained in S. cerevisiae suggest that the two ABCDs, which have been identified in this organism, form a heterodimer, which actually transports acyl coenzyme A esters across the peroxisomal membrane. In mammals, four ABCDs have been identified, of which one [adrenoleukodystrophy protein (ALDP)] has been implicated in the transport of the coenzyme A esters of very-long-chain fatty acids. Mutations in the gene (ABCD1) encoding ALDP are the cause of a severe X-linked disease, called X-linked adrenoleukodystrophy. The availability of mutant mice in which Abcd1, Abcd2, or Abcd3 have been disrupted will help to resolve the true role of the peroxisomal half-ABC transporters.

In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.

Peroxisomes are essential organelles of eukaryotic origin, ubiquitously distributed in cells and organisms, playing key roles in lipid and antioxidant metabolism. Loss or malfunction of peroxisomes causes more than 20 fatal inherited conditions. We have created a peroxisomal database (http://www.peroxisomeDB.org) that includes the complete peroxisomal proteome of Homo sapiens and Saccharomyces cerevisiae, by gathering, updating and integrating the available genetic and functional information on peroxisomal genes. PeroxisomeDB is structured in interrelated sections 'Genes', 'Functions', 'Metabolic pathways' and 'Diseases', that include hyperlinks to selected features of NCBI, ENSEMBL and UCSC databases. We have designed graphical depictions of the main peroxisomal metabolic routes and have included updated flow charts for diagnosis. Precomputed BLAST, PSI-BLAST, multiple sequence alignment (MUSCLE) and phylogenetic trees are provided to assist in direct multispecies comparison to study evolutionary conserved functions and pathways. Highlights of the PeroxisomeDB include new tools developed for facilitating (i) identification of novel peroxisomal proteins, by means of identifying proteins carrying peroxisome targeting signal (PTS) motifs, (ii) detection of peroxisomes in silico, particularly useful for screening the deluge of newly sequenced genomes. PeroxisomeDB should contribute to the systematic characterization of the peroxisomal proteome and facilitate system biology approaches on the organelle.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched chain fatty acid, which is a constituent of the human diet. The presence of the 3-methyl group of phytanic acid prevents degradation by beta-oxidation. Instead, the terminal carboxyl group is first removed by alpha-oxidation. The mechanism of the alpha-oxidation pathway and the enzymes involved are described in this review.

Peroxisomal disorders are a group of inherited diseases in man in which either peroxisome biogenesis or one or more peroxisomal functions are impaired. The peroxisomal disorders identified to date are usually classified in two groups including: (1) the disorders of peroxisome biogenesis, and (2) the single peroxisomal enzyme deficiencies. This review is focused on the second group of disorders, which currently includes ten different diseases in which the mutant gene affects a protein involved in one of the following peroxisomal functions: (1) ether phospholipid (plasmalogen) biosynthesis; (2) fatty acid beta-oxidation; (3) peroxisomal alpha-oxidation; (4) glyoxylate detoxification, and (5) H2O2 metabolism.

Peroxisomes contain specific transporter proteins required for the translocation of various metabolites across its membrane. The presence of several members of the ATP-binding cassette (ABC) transporter family is well established, and the characterization of transporters for adenine nucleotides and (pyro)phosphate in the peroxisomal membrane has been described recently. Previously published data strongly suggest the presence of additional transporters that facilitate the translocation of reducing equivalents and acetyl-units across the peroxisomal membrane. In this paper, we demonstrate the presence of transporter activity for 2-ketoglutarate and isocitrate in the peroxisomal membrane, by functional reconstitution of bovine kidney peroxisomal membrane protein in proteoliposomes. This transporter activity is assumed to be required to sustain the activity of intraperoxisomal isocitrate-dehydrogenase, which is involved in the regeneration of NADPH in the peroxisomal matrix.

Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.

Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.

Proteomic analysis of mouse kidney peroxisomes resulted in the identification of a novel nudix hydrolase designated RP2p, which is encoded by the D7RP2e gene. RP2p consists of 357 amino acids and contains two conserved domains: a nudix hydrolase domain and a CoA-binding domain. In addition, a PTS (peroxisomal targeting signal) type 1 (Ala-His-Leu) was found at the C-terminus. Analysis of the enzyme characteristics revealed that RP2p is a CoA diphosphatase with activity towards CoA, oxidized CoA and a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. The enzymatic properties of RP2p indicate that at low substrate concentrations medium and long-chain fatty-acyl-CoA esters are the primary substrates. Enzyme activity was optimal at pH 9 or above, and required the presence of Mg2+ or Mn2+ ions. Subcellular fractionation studies revealed that all CoA diphosphatase activity in mouse kidney is restricted to peroxisomes.

In this review, we describe the current state of knowledge about the biochemistry of mammalian peroxisomes, especially human peroxisomes. The identification and characterization of yeast mutants defective either in the biogenesis of peroxisomes or in one of its metabolic functions, notably fatty acid beta-oxidation, combined with the recognition of a group of genetic diseases in man, wherein these processes are also defective, have provided new insights in all aspects of peroxisomes. As a result of these and other studies, the indispensable role of peroxisomes in multiple metabolic pathways has been clarified, and many of the enzymes involved in these pathways have been characterized, purified, and cloned. One aspect of peroxisomes, which has remained ill defined, is the transport of metabolites across the peroxisomal membrane. Although it is clear that mammalian peroxisomes under in vivo conditions are closed structures, which require the active presence of metabolite transporter proteins, much remains to be learned about the permeability properties of mammalian peroxisomes and the role of the four half ATP-binding cassette (ABC) transporters therein.

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.

Pex19p is a protein required for the peroxisomal membrane synthesis. The 70-kDa peroxisomal membrane protein (PMP70) is synthesized on free cytosolic ribosomes and then inserted posttranslationally into peroxisomal membranes. Pex19p has been shown to play an important role in this process. Using an in vitro translation system, we investigated the role of Pex19p as a chaperone and identified the regions of PMP70 required for the interaction with Pex19p. When PMP70 was translated in the presence of purified Pex19p, a large part of PMP70 existed as soluble form and was co-immunoprecipitated with Pex19p. However, in the absence of Pex19p, PMP70 formed aggregates during translation. To identify the regions that interact with Pex19p, various truncated PMP70 were translated in the presence of Pex19p and subjected to co-immunoprecipitation. The interaction was markedly reduced by the deletion of the NH(2)-terminal 61 amino acids or the region around TMD6. Further, we expressed these deletion constructs of PMP70 in fusion with the green fluorescent protein in CHO cells. Fusion proteins lacking these Pex19p binding sites did not display any peroxisomal localization. These results suggest that Pex19p binds to PMP70 co-translationally and keeps PMP70 as a proper conformation for the localization to peroxisome.

It is now well established that the peroxisomal membrane is not freely permeable to small molecules in vivo, which implies the existence of metabolite transporters in the peroxisomal membrane. A few putative peroxisomal metabolite transporters have indeed been identified, but the function of these proteins has remained largely unresolved so far. The only peroxisomal transporter characterized to a significant extent is the adenine nucleotide transporter, which is presumably required to sustain the activity of the intraperoxisomal very-long-chain-acyl-CoA synthetase. In addition to AMP, this acyl-CoA synthetase also produces pyrophosphate, which must be exported from the peroxisome. In the present study, we demonstrate that the peroxisomal membrane contains a transporter activity that facilitates the passage of phosphate and possibly pyrophosphate across the peroxisomal membrane. By reconstitution of peroxisomal membrane proteins in proteoliposomes, some kinetic parameters of the transporter could be established in vitro. The transporter can be distinguished from the mitochondrial phosphate transporter by its different sensitivity to inhibitors.

Overexpression of alpha-methylacyl-CoA racemase (AMACR), an enzyme involved in branched chain fatty acid beta-oxidation, in prostate cancer has been reported. Here, we report that an enzyme downstream from AMACR in the peroxisomal branched chain fatty acid beta-oxidation pathway-D-bifunctional protein (DBP)-is also upregulated in prostate cancer at both mRNA and protein levels, accompanied by increased enzymatic activity. Furthermore, our data suggest that pristanoyl-CoA oxidase (ACOX3), which is expressed at extremely low level in other human organs studied including the liver, might contribute significantly to peroxisomal branched chain fatty acid beta-oxidation in human prostate tissue and some prostate cancer cell lines. In contrast to these results for peroxisomal enzymes, no significant expression changes of mitochondrial fatty acid beta-oxidation enzymes were observed in prostate cancer tissues through comprehensive quantitative RT-PCR screening. These data for the first time provide evidence for the selective over-activation of peroxisomal branched chain fatty acid beta-oxidation in prostate cancer, emphasizing a new metabolic change during prostate oncogenesis., (Copyright 2004 Wiley-Liss, Inc.)

We present a relatively mild case of peroxisomal D-bifunctional protein deficiency with inconsistent screening results in plasma for peroxisomal disorders.

Peroxisomal biogenesis defects include a number of severe neurodevelopmental disorders, among which infantile Refsum disease (IRD) occupies the mildest end of the spectrum. Although high docosahexaenoic acid (DHA) and low phytanic acid diets can correct some of the biochemical defects, they have not consistently altered the progressive course of the disease. We carried out orthotopic liver transplantation (OLT) in a mildly symptomatic 6-month-old infant who was a sibling of a severely neurologically impaired older sister. After transplantation the clinical course of this young child appeared much improved by comparison to her older sister. She walked alone at 4 years, had acceptable social interaction and had a noticeable recovery of audition. After transplantation her biochemical parameters were significantly improved: phytanic acid and very long-chain fatty acid (VLCFA) serum concentrations decreased. Abnormal bile acids disappeared from plasma. Although the OLT did not result in a cure of the disorder, the clinical and biochemical results suggest that OLT should be considered in mildly symptomatic patients.

Peroxisomes catalyse a large variety of different cellular functions of which most have to do with lipid metabolism. This paper deals with the role of peroxisomes in three key pathways of lipid metabolism, including: (1) etherphospholipid biosynthesis, (2) fatty acid beta-oxidation, and (3) fatty acid alpha-oxidation. Apart from a brief description of the peroxisomal enzymes involved in each of these pathways, the interaction between peroxisomes and other subcellular organelles, notably microsomes and peroxisomes, will be discussed. Finally, the current state of knowledge with respect to the different disorders of peroxisomal lipid metabolism will be described.

Peroxisomes have a central function in lipid metabolism, including the beta-oxidation of various fatty acids. The products and substrates involved in the beta-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH(per) 8.2), while the cytosolic pH was neutral (pH(cyt) 7.0). No Delta pH was present in ant1 Delta cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 Delta cells, while the peroxisomal pH remained 8.1+/-0.2 in pxa2 Delta cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the beta-oxidation of medium chain fatty acids.

Cysteinyl leukotrienes (LTs) are potent lipid mediators which are predominantly eliminated via bile. Their metabolic inactivation and degradation proceeds by beta-oxidation. However, although bile is the optimal material for analysis of LTs in man, only very sparse data on bile LT concentration under normal or pathophysiological conditions exist. The aim of the present study was to present for the first time a complete profile of endogenous LTs in human bile and to investigate the importance of bile LT analysis in peroxisomal and mitochondrial beta-oxidation deficiency. Cysteinyl LTs and their oxidation metabolites were analysed after HPLC separation by specific immunoassays or gas chromatography-mass spectrometry. Under physiological conditions, LTs are found in human bile (n = 8) in the nanomolar range with LTD4 predominating, whereas the other LTs were present in similar amounts. In bile of a patient with a peroxisome biogenesis disorder (Zellweger syndrome, ZS) LTE(4) was found to be slightly increased, whereas both omega-oxidation metabolites of LTE4, omega-hydroxy-LTE4 and omega-carboxy-LTE4, were highly increased (about 12-18 times). The beta-oxidation metabolite omega-carboxy-tetranor-LTE3 was below the detection limit (< 0.1 nmol/l; controls 1.4 +/- 1.2 nmol/l). This abnormal profile demonstrates an impaired degradation of LTs in ZS. In contrast, patients with X-linked adrenoleukodystrophy (X-ALD), medium-chain acyl CoA dehydrogenase deficiency (MCAD) as well as very long-chain acyl CoA dehydrogenase deficiency (VLCAD) did not show any differences in their biliary profile of LTs compared to controls. Increased levels of the biologically active cysteinyl LTs in the bile of patients with ZS might be of pathophysiological significance in the course of the disease, e.g. contributing to liver injury. In addition, our data confirm that the beta-oxidation of cysteinyl LTs in vivo occurs in peroxisomes and not in mitochondria., (Copyright 2004 Elsevier B.V.)