Mini Review Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders RJA Wanders and HR Waterham Department of Pediatrics, Academic Medical Centre, Emma Children’s Hospital, University of Amsterdam, and Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands Key words: fatty acids – genetics – inborn errors – peroxisome biogenesis – peroxisomes Corresponding author: Prof. Dr Ronald J. A. Wanders, Lab Genetic Metabolic Diseases, F0-224, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: þ31 20 5665958; fax: þ31 20 6962596; e-mail: [email protected] Received 7 May 2004, revised and accepted for publication 22 June 2004 Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 2004: 67: 107–133. # Blackwell Munksgaard, 2004 The peroxisomal disorders represent a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided into two subgroups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD group is comprised of four different disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum’s disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD the less severe disorders. Studies in the late 1980s had already shown that the PBD group is genetically heterogeneous with at least 12 distinct genetic groups as concluded from complementation studies. Thanks to the much improved knowledge about peroxisome biogenesis notably in yeasts and the successful extrapolation of this knowledge to humans, the genes responsible for all these complementation groups have been identified making molecular diagnosis of PBD patients feasible now. It is the purpose of this review to describe the current stage of knowledge about the clinical, biochemical, cellular, and molecular aspects of PBDs, and to provide guidelines for the post- and prenatal diagnosis of PBDs. Less progress has been made with respect to the pathophysiology and therapy of PBDs. The increasing availability of mouse models for these disorders is a major step forward in this respect. Zellweger syndrome (ZS) is the prototype of the group of peroxisomal disorders and was first described in the 1960s in two pairs of sibs, show- ing a series of abnormalities including craniofa- cial, hepatological, ocular, and skeletal aberrations. At about the same time, De Duve and coworkers performed systematic studies in which rat liver homogenates were subjected to differential and density gradient centrifugation. These studies led to the identification of a new organelle containing a number of H 2 O 2 -generating oxidases and catalase which decomposes H 2 O 2 to O 2 and H 2 O. The con- nection between ZS and peroxisomes first became apparent in 1973 when Goldfischer et al. (1) reported the absence of morphologically identifiable perox- isomes in hepatocytes and kidney tubule cells of Zellweger patients. At that time, however, vir- tually nothing was known about peroxisomes and it took another 10 years before the true sig- nificance of peroxisomes for human physiology started to become clear, thanks to two key obser- vations in Zellweger patients. First, Brown et al. (2) discovered distinct abnormalities in the fatty acid profile of plasma from Zellweger patients with markedly elevated levels of the very-long- chain fatty acids (VLCFAs) C24:0 and C26:0, whereas normal levels were found for the other fatty acids including long-chain fatty acids like palmitic, oleic, and linoleic acid. At that time, peroxisomes were already known to contain a fatty acid beta-oxidation system, just like mito- chondria, but the function of this system had remained obscure. The findings by Brown et al. (2) suggested that peroxisomes are the site of beta-oxidation of VLCFAs, which was soon established experimentally (3). The second major Clin Genet 2004: 67: 107–133 Copyright # Blackwell Munksgaard 2004 Printed in Singapore. All rights reserved CLINICAL GENETICS doi: 10.1111/j.1399-0004.2004.00329.x 107
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UnknownRJA Wanders and HR Waterham
Department of Pediatrics, Academic Medical Centre, Emma Children’s Hospital, University of Amsterdam, and Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands
Key words: fatty acids – genetics – inborn errors – peroxisome biogenesis – peroxisomes
Corresponding author: Prof. Dr Ronald J. A. Wanders, Lab Genetic Metabolic Diseases, F0-224, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: þ31 205665958; fax: þ31 206962596; e-mail: [email protected]
Received 7 May 2004, revised and accepted for publication 22 June 2004
Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 2004: 67: 107–133. # Blackwell Munksgaard, 2004
The peroxisomal disorders represent a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided into two subgroups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD group is comprised of four different disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum’s disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD the less severe disorders. Studies in the late 1980s had already shown that the PBD group is genetically heterogeneous with at least 12 distinct genetic groups as concluded from complementation studies. Thanks to the much improved knowledge about peroxisome biogenesis notably in yeasts and the successful extrapolation of this knowledge to humans, the genes responsible for all these complementation groups have been identified making molecular diagnosis of PBD patients feasible now. It is the purpose of this review to describe the current stage of knowledge about the clinical, biochemical, cellular, and molecular aspects of PBDs, and to provide guidelines for the post- and prenatal diagnosis of PBDs. Less progress has been made with respect to the pathophysiology and therapy of PBDs. The increasing availability of mouse models for these disorders is a major step forward in this respect.
Zellweger syndrome (ZS) is the prototype of the group of peroxisomal disorders and was first described in the 1960s in two pairs of sibs, show- ing a series of abnormalities including craniofa- cial, hepatological, ocular, and skeletal aberrations. At about the same time, De Duve and coworkers performed systematic studies in which rat liver homogenates were subjected to differential and density gradient centrifugation. These studies led to the identification of a new organelle containing a number of H2O2-generating oxidases and catalase which decomposes H2O2 to O2 and H2O. The con- nection between ZS and peroxisomes first became apparent in 1973whenGoldfischer et al. (1) reported the absence of morphologically identifiable perox- isomes in hepatocytes and kidney tubule cells of Zellweger patients. At that time, however, vir- tually nothing was known about peroxisomes
and it took another 10 years before the true sig- nificance of peroxisomes for human physiology started to become clear, thanks to two key obser- vations in Zellweger patients. First, Brown et al. (2) discovered distinct abnormalities in the fatty acid profile of plasma from Zellweger patients with markedly elevated levels of the very-long- chain fatty acids (VLCFAs) C24:0 and C26:0, whereas normal levels were found for the other fatty acids including long-chain fatty acids like palmitic, oleic, and linoleic acid. At that time, peroxisomes were already known to contain a fatty acid beta-oxidation system, just like mito- chondria, but the function of this system had remained obscure. The findings by Brown et al. (2) suggested that peroxisomes are the site of beta-oxidation of VLCFAs, which was soon established experimentally (3). The second major
Clin Genet 2004: 67: 107–133 Copyright # Blackwell Munksgaard 2004
Printed in Singapore. All rights reserved CLINICALGENETICS
doi: 10.1111/j.1399-0004.2004.00329.x
107
discovery demonstrating the crucial role of per- oxisomes in humans appeared 1 year later when Heymans et al. (4) reported the deficiency of plas- malogens, a special type of phospholipids belong- ing to the group of ether-linked phospholipids, in tissues from Zellweger patients. Since then, much has been learned about the metabolic role of peroxisomes and many different functions of per- oxisomes have been identified. In addition, many of the enzymes involved in the different metabolic pathways within peroxisomes have been charac- terized, purified, and their respective cDNAs and genes cloned. Parallel to this work, the essential details of peroxisome biogenesis have been worked out and many of the genes, coding for proteins essential for peroxisome biogenesis, have been identified. Thanks to this explosion of new information, enormous progress has been made with respect to the identification of new peroxisomal disorders followed by resolution of the underlying defects. At present, the group of peroxisomal disorders comprises 17 well-defined disorders, which are subdivided into two groups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. This review is focused on the first group of disorders, the PBDs (Table 1), and we will begin by discussing what is known about the different PBDs.
The peroxisome biogenesis disorders: a clinically and genetically heterogeneous group of disorders
The PBD group is comprised of four different disorders including ZS, neonatal adrenoleuko- dystrophy (NALD), infantile Refsum’s disease (IRD), and rhizomelic chondrodysplasia punc- tata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are nowadays usually referred to as ‘the Zellweger spectrum’ with ZS being the most severe and NALD and IRD less severe disorders. ZS is generally considered as the prototype of the PBD group. ZS is dominated by: (i) the typical craniofacial dysmorphism including a high forehead, large anterior fontanel, hypo- plastic supraorbital ridges, epicanthal folds, and deformed earlobes, and (ii) profound neurolog-
ical abnormalities. ZS children show severe psy- chomotor retardation, profound hypotonia, neonatal seizures, glaucoma, retinal degenera- tion, and impaired hearing. There is usually calcific stippling of the epiphyses and small renal cysts. Brain abnormalities in ZS include not only cortical dysplasia and neuronal heteroto- pia but also regressive changes. There is dysmyelin- ation rather than demyelination. Patients with NALD have hypotonia and seizures, may have polymicrogyria, progressive white matter disease, and usually die in late infancy. Patients with IRD may have external features reminiscent of ZS but do not show disordered neuronal migration and no progressive white matter disease. Their cogni- tive and motor development varies between severe global handicaps and moderate learning disabilities with deafness and visual impairment due to retinopathy. Their survival is variable. Most patients with IRD reach childhood and some even reach adulthood. Clinical distinction between the different PBD phenotypes is not very well defined. Common to all three are liver dis- ease, variable neurodevelopmental delay, retino- pathy, and perceptive deafness with onset in the first months of life. RCDP is clinically quite different from ZS,
NALD, and IRD and characterized by a dis- proportionally short stature primarily affecting the proximal parts of the extremities, typical facial appearance, including a broad nasal bridge, epicanthus, high arched palate, dysplastic external ears, micrognathia, congenital contractures, char- acteristic ocular involvement, dwarfism, and severe mental retardation with spasticity. Most RCDP patients die in the first decade of life. ZS, NALD, IRD, and RCDP have been found
to be genetically heterogeneous as concluded from complementation studies as discussed later in this review. The molecular defects underlying these different complementation groups (CGs) have been resolved in recent years. Two different strategies have been very rewarding in the iden- tification of these mutant genes, which includes (i) homology probing, making use of the infor- mation from different yeast mutants, and (ii) functional complementation analysis based on the generation of peroxisome-deficient Chinese
Table 1. The peroxisome biogenesis disorders
Number Disorder Abbreviation Protein involved Gene Chromosome MIM
1 Zellweger syndrome ZS Peroxins PEX-genes Multiple loci 214100 2 Neonatal adrenoleukodystrophy NALD Peroxins PEX-genes Multiple loci 214110 3 Infantile Refsum’s disease IRD Peroxins PEX-genes Multiple loci 202370 4 Rhizomelic chondrodysplasia
punctata type 1 RCDP Type 1 Pex7p PEX7 6q21–q22 215100
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hamster ovary (CHO) cells. We will proceed by describing the current stage of knowledge about peroxisome biogenesis.
Peroxisome biogenisis: general aspects
Peroxisomal proteins are all encoded by nuclear genes and translated on free polyribosomes as first shown for urate oxidase and catalase, two peroxisomal matrix proteins, by Goldmann and Blobel (5), and Robbi and Lazarow (6), respect- ively. Later studies have shown the same for peroxisomal membrane proteins (PMPs) (7). After synthesis on free polyribosomes, the newly made peroxisomal proteins are targeted to per- oxisomes and then imported into pre-existing peroxisomes post-translationally, which implies that synthesis and import are sequential rather than simultaneous processes. In this way, peroxi- somes get bigger which requires recruitment of phospholipids most likely from the endoplasmic reticulum (ER) to be incorporated into the peroxisomal membrane. Growth of peroxisomes may continue until a critical size is reached after which peroxisomes divide into two daughter per-
oxisomes that can then undergo the same cycle of events (Fig. 1a). The import of peroxisomal matrix and mem-
brane proteins into peroxisomes is a multistep process involving recognition of the cargo pro- tein by a receptor in the cytosol, docking of the receptor–cargo complex at the peroxisomal mem- brane, translocation across the membrane, cargo release into the organelle, and receptor recycling. Correct targeting of peroxisomal matrix proteins is achieved via cis-acting sequences present in the primary peptide sequences, which are called per- oxisomal targeting signals (PTSs). Most matrix proteins are equipped with a PTS type 1 (PTS1), which is a C-terminal serine-lysine-leucine- COOH (SKL) tripeptide, or a conservative vari- ant thereof, like SHL in D-aminoacid oxidase, AKL in sterol carrier protein 2 (SCP2), etc (Table 2). A few matrix proteins are targeted via a different signal named PTS2, which is a 9-amino acid sequence located near the N-terminus with the amino acids in positions 1, 2, 8, and 9 being most important. The consensus PTS2 is R/K-L/V/ I-XXXXX-H/Q-A/L in which X is any amino acid. The PTS1 and PTS2 receptors have been
Fig. 1. Original (a) and modified (b) model for peroxisome biogenesis. (a) The original growth-and-division model proposed by Lazarow and Fujiki (139) in which peroxisomes were thought to be autonomous organelles, which could not form de novo. (b) The modified model of Lazarow and Fujiki with peroxisomes now envisaged as semiautonomous organelles with the capacity to form de novo.
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cloned and characterized from different species. The former, Pex5p, is a tetratricopeptide (TPR) repeat protein, whereas the latter, Pex7p, is a WD40 repeat protein, to be discussed later. Similar to matrix proteins, PMPs are synthe-
sized on free cytosolic ribosomes and targeted to the organelle by cis-acting targeting sequences (mPTS). In contrast to the simple PTS1 and PTS2 sequences found in matrix proteins, PMPs are directed to peroxisomes via, as yet, less well-defined targeting signals, to be discussed later.
Peroxisome biogenesis: de novo formation of peroxisomes or not?
As discussed above, peroxisome biogenesis resembles that of mitochondria and chloroplasts, which is true although the details are entirely different. Indeed, protein translocation into per- oxisomes differs markedly from that in mitochon- dria which threads unfolded polypeptide chains through a narrow channel, whereas peroxisomes can import folded and homo-oligomeric proteins
(8), hetero-oligomers (9, 10), and even 4–9-nm gold beads (11). The transport of such large complexes somewhat resembles protein transport into the nucleus, but no such thing as a structure resembling the nuclear pore complex has ever been observed in the peroxisomal membrane. The concept that peroxisomes multiply by
growth and division of pre-existing peroxisomes would make peroxisomes belong to the group of autonomousorganelleswithmitochondria,chloro- plasts, and the endoplasmic reticulum as repre- sentatives. This would imply that peroxisomes cannot form de novo. Several experimental obser- vations have been done suggesting that peroxi- somes can form de novo, however. One of the main arguments in favor of de novo biogenesis of peroxisomes has been that cells, mutated in PEX3, PEX16, or PEX19, show no peroxisomal membrane structures (ghosts), whereas reintro- duction of a wild-type copy of the mutant gene restores peroxisome formation. These findings have been interpreted as evidence for de novo synthesis of peroxisomes from some endomem- brane compartment such as the ER (Fig. 1b).
Table 2. List of bona fide peroxisomal (enzyme) proteins from humans and their PTS1 or PTS2 sequences
Peroxisomal function (Enzyme) protein PTS1/PTS2 Targeting sequence
Fatty acid b-oxidation Acyl-CoA oxidase 1 (straight chain) PTS1 –SKL Acyl-CoA oxidase 2 (branched chain) PTS1 –SKL Acyl-CoA oxidase 3 (pristanoyl-CoA) PTS1 –SKL L-bifunctional protein PTS1 –SKL D-bifunctional protein PTS1 –AKL 3-ketothiolase (straight chain) PTS2 –RLQVVLGHL 3-ketothiolase (branched chain) PTS1 –AKL 2-methylacyl-CoA racemase PTS1 (þMTS) –(K)ASL Carnitine acetyltransferase PTS1 –AKL Carnitine octanoyltransferase PTS1 –THL Acyl-CoA thioesterase PTS1 –SKL Bile acid-CoA: taurine/glycine conjugating enzyme PTS1 –SQL 2,4-dienoylCoA reductase PTS1 –AKL D2,D3-enoylCoA isomerase PTS1 –SKL D3,5, D2,4-dienoylCoA isomerase PTS1 –SKL Very-long-chain acyl-CoA synthetase (VLACS) PTS1 –LKL
Fatty acid a-oxidation PhytanoylCoA hydroxylase PTS2 –RLQIVLGHL 2-hydroxyphytanoylCoA lyase PTS1 –(R)SNM
Etherphospholipid biosynthesis Dihydroxyacetonephosphate acyltransferase PTS1 –AKL Alkyldihydroxyacetonephosphate synthase PTS2 –RLVLSGHL
Glyoxylate detoxification Alanine glyoxylate aminotransferase PTS1 –KKL Pipecolic acid degradation L-pipecolate oxidase PTS1 –AHL H2O2 metabolism Catalase PTS1 –(K)ANL
Peroxiredoxin V PTS1 –SQL Sterol carrier protein 2 PTS1 –AKL D-aspartate oxidase PTS1 –(K)SNL D-amino acid oxidase PTS1 –SHL Hydroxyacid oxidase 3 PTS1 –SRL Hydroxyacid oxidase 2 PTS1 –SRL Hydroxyacid oxidase 1 (glycolate oxidase) PTS1 –SKL
Others 3-hydroxy-3-methyl glutarylCoA lyase PTS1 (þMTS) –CKL MalonylCoA decarboxylase PTS1 (þMTS) –SKL Isocitrate dehydrogenase PTS1 –AKL
PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; MTS, mitochondrial targeting signal. In the right hand column, the different targeting sequences are shown using the single letter code for the various amino acids.
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Based on studies in the yeasts Yarrowia lipoly- tica and Hansenula polymorpha, it has been pro- posed that peroxisomes can be formed from small pre-peroxisomal vesicles derived from the ER in a process dependent on COPI and COPII, two coat proteins involved in vesicle transport processes. Studies in human fibroblasts, however, have shown that peroxisome biogenesis occurs inde- pendent of COPI and COPII (12, 13). Further- more, studies by South et al. (14) in the yeast Saccharomyces cerevisiae suggest that protein traffic into the ER is not required to form peroxisomes. This was concluded from studies in which the protein entry into the ER was blocked by inactivation of the ER protein translocation factor, Sec61p, or its homolog, Ssh1p. These results argue against the ER as the site of de novo peroxisome formation. Furthermore, studies by Snyder et al. (15) and Hazra et al. (16) have provided compelling evidence against the dogma of the absence of peroxisomal structures in pex3D, pex16D, and pex19D mutants. Indeed, Snyder et al. (15) identified tiny peroxisomal vesicles and tubules in Pichia pastoris pex19D cells by deconvolu- tion microscopy using an antibody recognizing endogenous Pex3p. In addition, Hazra et al. (16) reported the identification of vesicular and tubular, torpedo-shaped peroxisomal structures in P. pastoris pex3D cells and characterized these by isopyknic and flotation centrifugation. The jury is still out on the origin of per-
oxisomes, however, as emphasized by several very recent studies. Firstly, Geuze et al. (17) recently presented evidence of the involvement of the ER in peroxisome formation in mouse dendritic cells using electron microscopy, immuno- cytochemistry, and three-dimensional image reconstruction of peroxisomes and associated com- partments. Additional support for the formation of peroxisomes from some endomembrane com- partment has also come from studies by Faber et al. (18) who have shown that an N-terminal fragment of Pex3p expressed in H. polymorpha is associated with vesicular membrane structures that also contain Pex14p. Furthermore, these structures appeared to have the potential to develop into functional peroxisomes after intro- duction of full-length PEX3 and arise from the nuclear membrane. In conclusion, it remains to be established whether there are indeed two parallel pathways for peroxisome formation, one from pre-existing peroxisomes and a second de novo pathway, which allows peroxisome formation from some endomembrane compartment such as the ER.
Peroxisome biogenesis: a closer look
The realization that a simple organism like baker’s yeast could be used to study peroxisome biogenesis and resolve the sorting and targeting of peroxisomal proteins to their correct destin- ation, the peroxisome, has had a tremendous impact and explains for a large part why the pur- suit of genes defect in PBD patients has been so fruitful in the last few years. The key to the application of genetics to the elucidation of the mechanism of peroxisome biogenesis and the identification of the proteins involved was the isolation of peroxisome-deficient mutants (pex mutants) from different yeast species and CHO mutants (19). Erdmann et al. (20) were the first to device a selection screen based on the notion that in yeast, peroxisomes are essential for growth on oleate. This follows logically from the fact that in yeast, fatty acids can only be oxidized in peroxisomes whereas in higher eukaryotes beta- oxidation can occur both in peroxisomes and in mitochondria. S. cerevisiae cells were sub- jected to chemical mutagenesis and grown first on glucose agar plates followed by replica plat- ing onto oleate agar plates to select for cells not growing on oleate (onu-mutants). Subsequently, cell fractionation studies were performed to eliminate mutants with no abnormalities in per- oxisome biogenesis but a defect in the fatty acid beta-oxidation system. This approach resulted in a total of 12 different mutants that turned out to be peroxisome deficient. Similar screens have been set up for a variety of different yeast species including P. pastoris, H. polymorpha, and Y. lipolytica. Additional screens and selections, based on other approaches, have also been set up which together has led to the generation of a large series of peroxisome biogenesis mutants. Subsequent complementation of these mutants using yeast genomic libraries has resulted in the identification of a large number of genes involved in peroxisome biogenesis. Initially, these new genes were all given different names even within the same species (i.e. PAF, PAS, PEB, PER, and PAY genes). To simplify matters, all of these genes have been renamed as PEX genes (PEX1, PEX2, PEX, etc) and the products of these genes are called peroxins (Pex1p, Pex2p, Pex3p, etc). The peroxins were agreed to include all proteins involved in peroxisome biogenesis inclusive of peroxisome matrix protein import, membrane biogenesis, peroxisome proliferation, and peroxi- some inheritance. In the original study of Erdmann et al. (20),
12 different S. cerevisiae mutants were identified in which peroxisome biogenesis was impaired.
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One by one the genes mutated in each of these so-called pas-mutants have been identified, of which the first one was described by Erdmann et al. in 1991 (21). The gene involved (PEX1) codes for a protein belonging to the family of triple A (AAA) ATPases, which are involved in the assembly, organization, and disassembly of protein complexes (22). The discovery of the first peroxisome biogenesis gene in S. cerevisiae was soon followed by reports from the same group describing the second (PEX3) (23) and third (PEX4) (24) S. cerevisiae PEX genes. In pex1D, pex3D, and pex4D cells, the import of PTS1 and PTS2 proteins is impaired, indicating that Pex1p, Pex3p, and Pex4p play an essential role in the import of matrix proteins. Later stud- ies revealed that these mutants are different if the import of PMPs is studied. Indeed, pex1D and pex4D cells are still able to assemble their PMPs into membranes, whereas pex3D cells lack this property. Studies by Hettema et al. (25) in a series of 19 S. cerevisiae mutants have shown that the import of PTS1 and/or PTS2 proteins is impaired in all mutants except one (pex11D), whereas PMP import is normal in all these mutants except for the pex3D and pex19D mutants. These data are in line with the notion that Pex3p and…
Department of Pediatrics, Academic Medical Centre, Emma Children’s Hospital, University of Amsterdam, and Department of Clinical Chemistry, Laboratory Genetic Metabolic Diseases, Amsterdam, The Netherlands
Key words: fatty acids – genetics – inborn errors – peroxisome biogenesis – peroxisomes
Corresponding author: Prof. Dr Ronald J. A. Wanders, Lab Genetic Metabolic Diseases, F0-224, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel.: þ31 205665958; fax: þ31 206962596; e-mail: [email protected]
Received 7 May 2004, revised and accepted for publication 22 June 2004
Wanders RJA, Waterham HR. Peroxisomal disorders I: biochemistry and genetics of peroxisome biogenesis disorders. Clin Genet 2004: 67: 107–133. # Blackwell Munksgaard, 2004
The peroxisomal disorders represent a group of genetic diseases in humans in which there is an impairment in one or more peroxisomal functions. The peroxisomal disorders are usually subdivided into two subgroups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. The PBD group is comprised of four different disorders including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum’s disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are usually referred to as the Zellweger spectrum with ZS being the most severe and NALD and IRD the less severe disorders. Studies in the late 1980s had already shown that the PBD group is genetically heterogeneous with at least 12 distinct genetic groups as concluded from complementation studies. Thanks to the much improved knowledge about peroxisome biogenesis notably in yeasts and the successful extrapolation of this knowledge to humans, the genes responsible for all these complementation groups have been identified making molecular diagnosis of PBD patients feasible now. It is the purpose of this review to describe the current stage of knowledge about the clinical, biochemical, cellular, and molecular aspects of PBDs, and to provide guidelines for the post- and prenatal diagnosis of PBDs. Less progress has been made with respect to the pathophysiology and therapy of PBDs. The increasing availability of mouse models for these disorders is a major step forward in this respect.
Zellweger syndrome (ZS) is the prototype of the group of peroxisomal disorders and was first described in the 1960s in two pairs of sibs, show- ing a series of abnormalities including craniofa- cial, hepatological, ocular, and skeletal aberrations. At about the same time, De Duve and coworkers performed systematic studies in which rat liver homogenates were subjected to differential and density gradient centrifugation. These studies led to the identification of a new organelle containing a number of H2O2-generating oxidases and catalase which decomposes H2O2 to O2 and H2O. The con- nection between ZS and peroxisomes first became apparent in 1973whenGoldfischer et al. (1) reported the absence of morphologically identifiable perox- isomes in hepatocytes and kidney tubule cells of Zellweger patients. At that time, however, vir- tually nothing was known about peroxisomes
and it took another 10 years before the true sig- nificance of peroxisomes for human physiology started to become clear, thanks to two key obser- vations in Zellweger patients. First, Brown et al. (2) discovered distinct abnormalities in the fatty acid profile of plasma from Zellweger patients with markedly elevated levels of the very-long- chain fatty acids (VLCFAs) C24:0 and C26:0, whereas normal levels were found for the other fatty acids including long-chain fatty acids like palmitic, oleic, and linoleic acid. At that time, peroxisomes were already known to contain a fatty acid beta-oxidation system, just like mito- chondria, but the function of this system had remained obscure. The findings by Brown et al. (2) suggested that peroxisomes are the site of beta-oxidation of VLCFAs, which was soon established experimentally (3). The second major
Clin Genet 2004: 67: 107–133 Copyright # Blackwell Munksgaard 2004
Printed in Singapore. All rights reserved CLINICALGENETICS
doi: 10.1111/j.1399-0004.2004.00329.x
107
discovery demonstrating the crucial role of per- oxisomes in humans appeared 1 year later when Heymans et al. (4) reported the deficiency of plas- malogens, a special type of phospholipids belong- ing to the group of ether-linked phospholipids, in tissues from Zellweger patients. Since then, much has been learned about the metabolic role of peroxisomes and many different functions of per- oxisomes have been identified. In addition, many of the enzymes involved in the different metabolic pathways within peroxisomes have been charac- terized, purified, and their respective cDNAs and genes cloned. Parallel to this work, the essential details of peroxisome biogenesis have been worked out and many of the genes, coding for proteins essential for peroxisome biogenesis, have been identified. Thanks to this explosion of new information, enormous progress has been made with respect to the identification of new peroxisomal disorders followed by resolution of the underlying defects. At present, the group of peroxisomal disorders comprises 17 well-defined disorders, which are subdivided into two groups including (i) the peroxisome biogenesis disorders (PBDs) and (ii) the single peroxisomal (enzyme-) protein deficiencies. This review is focused on the first group of disorders, the PBDs (Table 1), and we will begin by discussing what is known about the different PBDs.
The peroxisome biogenesis disorders: a clinically and genetically heterogeneous group of disorders
The PBD group is comprised of four different disorders including ZS, neonatal adrenoleuko- dystrophy (NALD), infantile Refsum’s disease (IRD), and rhizomelic chondrodysplasia punc- tata (RCDP). ZS, NALD, and IRD are clearly distinct from RCDP and are nowadays usually referred to as ‘the Zellweger spectrum’ with ZS being the most severe and NALD and IRD less severe disorders. ZS is generally considered as the prototype of the PBD group. ZS is dominated by: (i) the typical craniofacial dysmorphism including a high forehead, large anterior fontanel, hypo- plastic supraorbital ridges, epicanthal folds, and deformed earlobes, and (ii) profound neurolog-
ical abnormalities. ZS children show severe psy- chomotor retardation, profound hypotonia, neonatal seizures, glaucoma, retinal degenera- tion, and impaired hearing. There is usually calcific stippling of the epiphyses and small renal cysts. Brain abnormalities in ZS include not only cortical dysplasia and neuronal heteroto- pia but also regressive changes. There is dysmyelin- ation rather than demyelination. Patients with NALD have hypotonia and seizures, may have polymicrogyria, progressive white matter disease, and usually die in late infancy. Patients with IRD may have external features reminiscent of ZS but do not show disordered neuronal migration and no progressive white matter disease. Their cogni- tive and motor development varies between severe global handicaps and moderate learning disabilities with deafness and visual impairment due to retinopathy. Their survival is variable. Most patients with IRD reach childhood and some even reach adulthood. Clinical distinction between the different PBD phenotypes is not very well defined. Common to all three are liver dis- ease, variable neurodevelopmental delay, retino- pathy, and perceptive deafness with onset in the first months of life. RCDP is clinically quite different from ZS,
NALD, and IRD and characterized by a dis- proportionally short stature primarily affecting the proximal parts of the extremities, typical facial appearance, including a broad nasal bridge, epicanthus, high arched palate, dysplastic external ears, micrognathia, congenital contractures, char- acteristic ocular involvement, dwarfism, and severe mental retardation with spasticity. Most RCDP patients die in the first decade of life. ZS, NALD, IRD, and RCDP have been found
to be genetically heterogeneous as concluded from complementation studies as discussed later in this review. The molecular defects underlying these different complementation groups (CGs) have been resolved in recent years. Two different strategies have been very rewarding in the iden- tification of these mutant genes, which includes (i) homology probing, making use of the infor- mation from different yeast mutants, and (ii) functional complementation analysis based on the generation of peroxisome-deficient Chinese
Table 1. The peroxisome biogenesis disorders
Number Disorder Abbreviation Protein involved Gene Chromosome MIM
1 Zellweger syndrome ZS Peroxins PEX-genes Multiple loci 214100 2 Neonatal adrenoleukodystrophy NALD Peroxins PEX-genes Multiple loci 214110 3 Infantile Refsum’s disease IRD Peroxins PEX-genes Multiple loci 202370 4 Rhizomelic chondrodysplasia
punctata type 1 RCDP Type 1 Pex7p PEX7 6q21–q22 215100
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hamster ovary (CHO) cells. We will proceed by describing the current stage of knowledge about peroxisome biogenesis.
Peroxisome biogenisis: general aspects
Peroxisomal proteins are all encoded by nuclear genes and translated on free polyribosomes as first shown for urate oxidase and catalase, two peroxisomal matrix proteins, by Goldmann and Blobel (5), and Robbi and Lazarow (6), respect- ively. Later studies have shown the same for peroxisomal membrane proteins (PMPs) (7). After synthesis on free polyribosomes, the newly made peroxisomal proteins are targeted to per- oxisomes and then imported into pre-existing peroxisomes post-translationally, which implies that synthesis and import are sequential rather than simultaneous processes. In this way, peroxi- somes get bigger which requires recruitment of phospholipids most likely from the endoplasmic reticulum (ER) to be incorporated into the peroxisomal membrane. Growth of peroxisomes may continue until a critical size is reached after which peroxisomes divide into two daughter per-
oxisomes that can then undergo the same cycle of events (Fig. 1a). The import of peroxisomal matrix and mem-
brane proteins into peroxisomes is a multistep process involving recognition of the cargo pro- tein by a receptor in the cytosol, docking of the receptor–cargo complex at the peroxisomal mem- brane, translocation across the membrane, cargo release into the organelle, and receptor recycling. Correct targeting of peroxisomal matrix proteins is achieved via cis-acting sequences present in the primary peptide sequences, which are called per- oxisomal targeting signals (PTSs). Most matrix proteins are equipped with a PTS type 1 (PTS1), which is a C-terminal serine-lysine-leucine- COOH (SKL) tripeptide, or a conservative vari- ant thereof, like SHL in D-aminoacid oxidase, AKL in sterol carrier protein 2 (SCP2), etc (Table 2). A few matrix proteins are targeted via a different signal named PTS2, which is a 9-amino acid sequence located near the N-terminus with the amino acids in positions 1, 2, 8, and 9 being most important. The consensus PTS2 is R/K-L/V/ I-XXXXX-H/Q-A/L in which X is any amino acid. The PTS1 and PTS2 receptors have been
Fig. 1. Original (a) and modified (b) model for peroxisome biogenesis. (a) The original growth-and-division model proposed by Lazarow and Fujiki (139) in which peroxisomes were thought to be autonomous organelles, which could not form de novo. (b) The modified model of Lazarow and Fujiki with peroxisomes now envisaged as semiautonomous organelles with the capacity to form de novo.
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cloned and characterized from different species. The former, Pex5p, is a tetratricopeptide (TPR) repeat protein, whereas the latter, Pex7p, is a WD40 repeat protein, to be discussed later. Similar to matrix proteins, PMPs are synthe-
sized on free cytosolic ribosomes and targeted to the organelle by cis-acting targeting sequences (mPTS). In contrast to the simple PTS1 and PTS2 sequences found in matrix proteins, PMPs are directed to peroxisomes via, as yet, less well-defined targeting signals, to be discussed later.
Peroxisome biogenesis: de novo formation of peroxisomes or not?
As discussed above, peroxisome biogenesis resembles that of mitochondria and chloroplasts, which is true although the details are entirely different. Indeed, protein translocation into per- oxisomes differs markedly from that in mitochon- dria which threads unfolded polypeptide chains through a narrow channel, whereas peroxisomes can import folded and homo-oligomeric proteins
(8), hetero-oligomers (9, 10), and even 4–9-nm gold beads (11). The transport of such large complexes somewhat resembles protein transport into the nucleus, but no such thing as a structure resembling the nuclear pore complex has ever been observed in the peroxisomal membrane. The concept that peroxisomes multiply by
growth and division of pre-existing peroxisomes would make peroxisomes belong to the group of autonomousorganelleswithmitochondria,chloro- plasts, and the endoplasmic reticulum as repre- sentatives. This would imply that peroxisomes cannot form de novo. Several experimental obser- vations have been done suggesting that peroxi- somes can form de novo, however. One of the main arguments in favor of de novo biogenesis of peroxisomes has been that cells, mutated in PEX3, PEX16, or PEX19, show no peroxisomal membrane structures (ghosts), whereas reintro- duction of a wild-type copy of the mutant gene restores peroxisome formation. These findings have been interpreted as evidence for de novo synthesis of peroxisomes from some endomem- brane compartment such as the ER (Fig. 1b).
Table 2. List of bona fide peroxisomal (enzyme) proteins from humans and their PTS1 or PTS2 sequences
Peroxisomal function (Enzyme) protein PTS1/PTS2 Targeting sequence
Fatty acid b-oxidation Acyl-CoA oxidase 1 (straight chain) PTS1 –SKL Acyl-CoA oxidase 2 (branched chain) PTS1 –SKL Acyl-CoA oxidase 3 (pristanoyl-CoA) PTS1 –SKL L-bifunctional protein PTS1 –SKL D-bifunctional protein PTS1 –AKL 3-ketothiolase (straight chain) PTS2 –RLQVVLGHL 3-ketothiolase (branched chain) PTS1 –AKL 2-methylacyl-CoA racemase PTS1 (þMTS) –(K)ASL Carnitine acetyltransferase PTS1 –AKL Carnitine octanoyltransferase PTS1 –THL Acyl-CoA thioesterase PTS1 –SKL Bile acid-CoA: taurine/glycine conjugating enzyme PTS1 –SQL 2,4-dienoylCoA reductase PTS1 –AKL D2,D3-enoylCoA isomerase PTS1 –SKL D3,5, D2,4-dienoylCoA isomerase PTS1 –SKL Very-long-chain acyl-CoA synthetase (VLACS) PTS1 –LKL
Fatty acid a-oxidation PhytanoylCoA hydroxylase PTS2 –RLQIVLGHL 2-hydroxyphytanoylCoA lyase PTS1 –(R)SNM
Etherphospholipid biosynthesis Dihydroxyacetonephosphate acyltransferase PTS1 –AKL Alkyldihydroxyacetonephosphate synthase PTS2 –RLVLSGHL
Glyoxylate detoxification Alanine glyoxylate aminotransferase PTS1 –KKL Pipecolic acid degradation L-pipecolate oxidase PTS1 –AHL H2O2 metabolism Catalase PTS1 –(K)ANL
Peroxiredoxin V PTS1 –SQL Sterol carrier protein 2 PTS1 –AKL D-aspartate oxidase PTS1 –(K)SNL D-amino acid oxidase PTS1 –SHL Hydroxyacid oxidase 3 PTS1 –SRL Hydroxyacid oxidase 2 PTS1 –SRL Hydroxyacid oxidase 1 (glycolate oxidase) PTS1 –SKL
Others 3-hydroxy-3-methyl glutarylCoA lyase PTS1 (þMTS) –CKL MalonylCoA decarboxylase PTS1 (þMTS) –SKL Isocitrate dehydrogenase PTS1 –AKL
PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; MTS, mitochondrial targeting signal. In the right hand column, the different targeting sequences are shown using the single letter code for the various amino acids.
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Based on studies in the yeasts Yarrowia lipoly- tica and Hansenula polymorpha, it has been pro- posed that peroxisomes can be formed from small pre-peroxisomal vesicles derived from the ER in a process dependent on COPI and COPII, two coat proteins involved in vesicle transport processes. Studies in human fibroblasts, however, have shown that peroxisome biogenesis occurs inde- pendent of COPI and COPII (12, 13). Further- more, studies by South et al. (14) in the yeast Saccharomyces cerevisiae suggest that protein traffic into the ER is not required to form peroxisomes. This was concluded from studies in which the protein entry into the ER was blocked by inactivation of the ER protein translocation factor, Sec61p, or its homolog, Ssh1p. These results argue against the ER as the site of de novo peroxisome formation. Furthermore, studies by Snyder et al. (15) and Hazra et al. (16) have provided compelling evidence against the dogma of the absence of peroxisomal structures in pex3D, pex16D, and pex19D mutants. Indeed, Snyder et al. (15) identified tiny peroxisomal vesicles and tubules in Pichia pastoris pex19D cells by deconvolu- tion microscopy using an antibody recognizing endogenous Pex3p. In addition, Hazra et al. (16) reported the identification of vesicular and tubular, torpedo-shaped peroxisomal structures in P. pastoris pex3D cells and characterized these by isopyknic and flotation centrifugation. The jury is still out on the origin of per-
oxisomes, however, as emphasized by several very recent studies. Firstly, Geuze et al. (17) recently presented evidence of the involvement of the ER in peroxisome formation in mouse dendritic cells using electron microscopy, immuno- cytochemistry, and three-dimensional image reconstruction of peroxisomes and associated com- partments. Additional support for the formation of peroxisomes from some endomembrane com- partment has also come from studies by Faber et al. (18) who have shown that an N-terminal fragment of Pex3p expressed in H. polymorpha is associated with vesicular membrane structures that also contain Pex14p. Furthermore, these structures appeared to have the potential to develop into functional peroxisomes after intro- duction of full-length PEX3 and arise from the nuclear membrane. In conclusion, it remains to be established whether there are indeed two parallel pathways for peroxisome formation, one from pre-existing peroxisomes and a second de novo pathway, which allows peroxisome formation from some endomembrane compartment such as the ER.
Peroxisome biogenesis: a closer look
The realization that a simple organism like baker’s yeast could be used to study peroxisome biogenesis and resolve the sorting and targeting of peroxisomal proteins to their correct destin- ation, the peroxisome, has had a tremendous impact and explains for a large part why the pur- suit of genes defect in PBD patients has been so fruitful in the last few years. The key to the application of genetics to the elucidation of the mechanism of peroxisome biogenesis and the identification of the proteins involved was the isolation of peroxisome-deficient mutants (pex mutants) from different yeast species and CHO mutants (19). Erdmann et al. (20) were the first to device a selection screen based on the notion that in yeast, peroxisomes are essential for growth on oleate. This follows logically from the fact that in yeast, fatty acids can only be oxidized in peroxisomes whereas in higher eukaryotes beta- oxidation can occur both in peroxisomes and in mitochondria. S. cerevisiae cells were sub- jected to chemical mutagenesis and grown first on glucose agar plates followed by replica plat- ing onto oleate agar plates to select for cells not growing on oleate (onu-mutants). Subsequently, cell fractionation studies were performed to eliminate mutants with no abnormalities in per- oxisome biogenesis but a defect in the fatty acid beta-oxidation system. This approach resulted in a total of 12 different mutants that turned out to be peroxisome deficient. Similar screens have been set up for a variety of different yeast species including P. pastoris, H. polymorpha, and Y. lipolytica. Additional screens and selections, based on other approaches, have also been set up which together has led to the generation of a large series of peroxisome biogenesis mutants. Subsequent complementation of these mutants using yeast genomic libraries has resulted in the identification of a large number of genes involved in peroxisome biogenesis. Initially, these new genes were all given different names even within the same species (i.e. PAF, PAS, PEB, PER, and PAY genes). To simplify matters, all of these genes have been renamed as PEX genes (PEX1, PEX2, PEX, etc) and the products of these genes are called peroxins (Pex1p, Pex2p, Pex3p, etc). The peroxins were agreed to include all proteins involved in peroxisome biogenesis inclusive of peroxisome matrix protein import, membrane biogenesis, peroxisome proliferation, and peroxi- some inheritance. In the original study of Erdmann et al. (20),
12 different S. cerevisiae mutants were identified in which peroxisome biogenesis was impaired.
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One by one the genes mutated in each of these so-called pas-mutants have been identified, of which the first one was described by Erdmann et al. in 1991 (21). The gene involved (PEX1) codes for a protein belonging to the family of triple A (AAA) ATPases, which are involved in the assembly, organization, and disassembly of protein complexes (22). The discovery of the first peroxisome biogenesis gene in S. cerevisiae was soon followed by reports from the same group describing the second (PEX3) (23) and third (PEX4) (24) S. cerevisiae PEX genes. In pex1D, pex3D, and pex4D cells, the import of PTS1 and PTS2 proteins is impaired, indicating that Pex1p, Pex3p, and Pex4p play an essential role in the import of matrix proteins. Later stud- ies revealed that these mutants are different if the import of PMPs is studied. Indeed, pex1D and pex4D cells are still able to assemble their PMPs into membranes, whereas pex3D cells lack this property. Studies by Hettema et al. (25) in a series of 19 S. cerevisiae mutants have shown that the import of PTS1 and/or PTS2 proteins is impaired in all mutants except one (pex11D), whereas PMP import is normal in all these mutants except for the pex3D and pex19D mutants. These data are in line with the notion that Pex3p and…
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