Targeted Deletion of the PEX2 Peroxisome Assembly Gene in Mice Provides a Model for Zellweger Syndrome, a Human Neuronal Migration Disorder

Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting.

Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar–lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.

     

Molecular basis of peroxisomal biogenesis disorders caused by defects in peroxisomal matrix protein import

Peroxisomal biogenesis disorders (PBDs) represent a spectrum of autosomal recessive metabolic disorders that are collectively characterized by abnormal peroxisome assembly and impaired peroxisomal function. The importance of this ubiquitous organelle for human health is highlighted by the fact that PBDs are multisystemic disorders that often cause death in early infancy. Peroxisomes contribute to central metabolic pathways. Most enzymes in the peroxisomal matrix are linked to lipid metabolism and detoxification of reactive oxygen species. Proper assembly of peroxisomes and thus also import of their enzymes relies on specific peroxisomal biogenesis factors, so called peroxins with PEX being the gene acronym. To date, 13 PEX genes are known to cause PBDs when mutated. Studies of the cellular and molecular defects in cells derived from PBD patients have significantly contributed to the understanding of the functional role of the corresponding peroxins in peroxisome assembly. In this review, we discuss recent data derived from both human cell culture as well as model organisms like yeasts and present an overview on the molecular mechanism underlying peroxisomal biogenesis disorders with emphasis on disorders caused by defects in the peroxisomal matrix protein import machinery. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.     

Peroxisome biogenesis disorders: Molecular basis for impaired peroxisomal membrane assembly: In metabolic functions and biogenesis of peroxisomes in health and disease

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease.     

An Inventory of Peroxisomal Proteins and Pathways in Drosophila melanogaster

Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). Although much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to Caenorhabditis elegans, Drosophila appears to only utilize the peroxisome targeting signal type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system.     

Peroxisome biogenesis disorders: genetics and cell biology

Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and rhizomelic chondrodysplasia punctata are progressive disorders characterized by loss of multiple peroxisomal metabolic functions. These diseases are inherited in an autosomal recessive manner, are caused by defects in the import of peroxisomal matrix proteins and are referred to as the peroxisome biogenesis disorders (PBDs). Recent studies have identified the PEX genes that are mutated in 11 of the 12 known complementation groups of PBD patients. This article reviews these advances in PBD genetics and discusses how studies of human PEX genes, their protein products and PBD cell lines are shaping current models of peroxisome biogenesis.     

PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G

Peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy are autosomal recessive diseases caused by defects in peroxisome assembly, for which 13 genotypes have been identified. Expression of the human peroxin Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in fibroblasts from PBDG-02, a patient with complementation group G (CG-G) ZS. Patient PBDG-02 carried a homozygous, inactivating mutation-a 97-bp deletion of nucleotide residues at positions 942-1038-resulting in a 32-amino-acid truncation and in a frameshift inducing both a 3-amino-acid substitution and a termination codon. Genomic PCR analysis revealed mutation of T-->G at eight bases upstream of the splicing site at the boundary of intron 10 and exon 11 of PEX3 gene, giving rise to a deletion of all of exon 11. When assessed by expression in a pex3 mutant of Chinese hamster ovary cells and the patient's fibroblasts, PBDG-02-derived PEX3 cDNA was found to be defective in peroxisome-restoring activity. These results provide evidence that PEX3 is a novel, pathogenic gene responsible for CG-G PBDs.     

Complementation in Zellweger syndrome: biochemical analysis of newly generated peroxisomes.

The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.     

PEX11 promotes peroxisome division independently of peroxisome metabolism

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.     

The peroxisomal AAA ATPase complex prevents pexophagy and development of peroxisome biogenesis disorders

Peroxisome biogenesis disorders (PBDs) are metabolic disorders caused by the loss of peroxisomes. The majority of PBDs result from mutation in one of 3 genes that encode for the peroxisomal AAA ATPase complex (AAA-complex) required for cycling PEX5 for peroxisomal matrix protein import. Mutations in these genes are thought to result in a defect in peroxisome assembly by preventing the import of matrix proteins. However, we show here that loss of the AAA-complex does not prevent matrix protein import, but instead causes an upregulation of peroxisome degradation by macroautophagy, or pexophagy. The loss of AAA-complex function in cells results in the accumulation of ubiquitinated PEX5 on the peroxisomal membrane that signals pexophagy. Inhibiting autophagy by genetic or pharmacological approaches rescues peroxisome number, protein import and function. Our findings suggest that the peroxisomal AAA-complex is required for peroxisome quality control, whereas its absence results in the selective degradation of the peroxisome. Thus the loss of peroxisomes in PBD patients with mutations in their peroxisomal AAA-complex is a result of increased pexophagy. Our study also provides a framework for the development of novel therapeutic treatments for PBDs.     

Peroxisome biogenesis and the role of protein import

Peroxisomes are metabolic organelles with enzymatic content that are found in virtually all cells and are involved in β-oxidation of fatty acids, hydrogen peroxide-based respiration and defence against oxidative stress. The steps of their biogenesis involves "peroxins", proteins encoded by PEX genes. Peroxins are involved in three key stages of peroxisome development: (1) import of peroxisomal membrane proteins; (2) import of peroxisomal matrix proteins and (3) peroxisome proliferation. Of these three areas, peroxisomal matrix-protein import is by far the best understood and accounts for most of the available published data on peroxisome biogenesis. Defects in peroxisome biogenesis result in peroxisome biogenesis disorders (PBDs), which although rare, have no known cure to-date. This review explores current understanding of each key area in peroxisome biogenesis, paying particular attention to the role of protein import.     

Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motility and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells.     

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H₂O₂ and β-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein–protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.     

Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants

In higher plants, peroxisomes accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. Recently, many factors regulating peroxisomal biogenesis, so-called PEX genes, have been identified not only in plants but also in yeasts and mammals. In the Arabidopsis genome, the presence of at least 22 PEX genes has been proposed. Here, we clarify the physiological functions of 18 PEX genes for peroxisomal biogenesis by analyzing transgenic Arabidopsis plants that suppressed the PEX gene expression using RNA interference. The results indicated that the function of these PEX genes could be divided into two groups. One group involves PEX1, PEX2, PEX4, PEX6, PEX10, PEX12 and PEX13 together with previously characterized PEX5, PEX7 and PEX14. Defects in these genes caused loss of peroxisomal function due to misdistribution of peroxisomal matrix proteins in the cytosol. Of these, the pex10 mutant showed pleiotropic phenotypes that were not observed in any other pex mutants. In contrast, reduced peroxisomal function of the second group, including PEX3, PEX11, PEX16 and PEX19, was induced by morphological changes of the peroxisomes. Cells of the pex16 mutant in particular possessed reduced numbers of large peroxisome(s) that contained unknown vesicles. These results provide experimental evidence indicating that all of these PEX genes play pivotal roles in regulating peroxisomal biogenesis. We conclude that PEX genes belonging to the former group are involved in regulating peroxisomal protein import, whereas those of the latter group are important in maintaining the structure of peroxisome.     

The surprising complexity of peroxisome biogenesis

Peroxisomes are small organelles with a single boundary membrane. All of their matrix proteins are nuclear-encoded, synthesized on free ribosomes in the cytosol, and post-translationally transported into the organelle. This may sound familiar, but in fact, peroxisome biogenesis is proving to be surprisingly unique. First, there are several classes of plant peroxisomes, each specialized for a different metabolic function and sequestering specific matrix enzymes. Second, although the mechanisms of peroxisomal protein import are conserved between the classes, multiple pathways of protein targeting and translocation have been defined. At least two different types of targeting signals direct proteins to the peroxisome matrix. The most common peroxisomal targeting signal is a tripeptide limited to the carboxyl terminus of the protein. Some peroxisomal proteins possess an amino-terminal signal which may be cleaved after import. Each targeting signal interacts with a different cytosolic receptor; other cytosolic factors or chaperones may also form a complex with the peroxisomal protein before it docks on the membrane. Peroxisomes have the unusual capacity to import proteins that are fully folded or assembled into oligomers. Although at least 20 proteins (mostly peroxins) are required for peroxisome biogenesis, the role of only a few of these have been determined. Future efforts will be directed towards an understanding of how these proteins interact and contribute to the complex process of protein import into peroxisomes.     

The peroxisome: still a mysterious organelle

More than half a century of research on peroxisomes has revealed unique features of this ubiquitous subcellular organelle, which have often been in disagreement with existing dogmas in cell biology. About 50 peroxisomal enzymes have so far been identified, which contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, and render peroxisomes indispensable for human health and development. It became obvious that peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. However, many aspects of peroxisome biology are still mysterious. This review addresses recent exciting discoveries on the biogenesis, formation and degradation of peroxisomes, on peroxisomal dynamics and division, as well as on the interaction and cross talk of peroxisomes with other subcellular compartments. Furthermore, recent advances on the role of peroxisomes in medicine and in the identification of novel peroxisomal proteins are discussed.     

Overexpression of a human and a fungal ABC transporter similarly suppresses the differentiation defects of a fungal peroxisomal mutant but introduces pleiotropic cellular effects

Among the peroxisome membrane proteins, some are required for peroxisome biogenesis (e.g. PEX2) while others are not, e.g. ABC (ATP-binding cassette) transporters. Unexpectedly, overproduction of the peroxisomal ABC transporter PMP70 was found to be able to restore peroxisome biogenesis in mammalian pex2 mutant cell lines. In the filamentous fungus Podospora anserina, pex2 mutations not only impair peroxisome biogenesis but also cause a precise cell differentiation defect. Here, we show that both defects are partially suppressed by expression of the human cDNA encoding PMP70. In addition, PMP70 expression causes new developmental defects, different from those induced by pex2 mutations. We also show that overexpression of the P. anserina pABC1 gene, which encodes a peroxisomal ABC transporter, leads to similar effects. Taken together, our results demonstrate that: (i) the genetic relationship between PEX2 and PMP70, initially observed in mammals, has been conserved through evolution; (ii) the cell differentiation defect observed in the P. anserina pex2 mutants is indeed linked to impairment in peroxisome biogenesis; and (iii) unexpected detrimental cellular defects result from overproduction of peroxisomal ABC transporters.     

Peroxisome morphology in pathology

Peroxisomes are remarkably dynamic and versatile organelles that are essential for human health and development. They respond to physiological changes in the cellular environment by adapting their morphology, number, enzyme content and metabolic functions accordingly. With the discovery of the first key peroxisomal morphology proteins, the investigation of peroxisomal shape, distribution and dynamics has become an exciting new field in cell biology and biomedical sciences because of its relation to organelle functionality and its impact on developmental and physiological processes. In this review, we summarize recent findings on peroxisome biology, dynamics and the modulation of peroxisome morphology, especially in mammals. Furthermore, we discuss the roles of peroxisome dynamics and morphology in cell pathology and present recent examples for alterations in peroxisome morphology under disease conditions. Besides defects in the peroxisomal morphology machinery, we also address peroxisome biogenesis disorders, alterations of peroxisome number during carcinogenesis and liver cirrhosis, and morphological alterations of peroxisomes during viral infection.     

Host peroxisomal properties are not restored to normal after treatment of visceral leishmaniasis with sodium antimony gluconate

Reason for post-kala-azar dermal leishmaniasis (PKDL) is yet to be established. Earlier it was observed that morphology and biochemical properties of host peroxisomes were impaired during Leishmania infection. As peroxisome is known to be involved in various metabolic pathways to monitor normal function of the host cells, it is essential that Leishmania-induced dysfunction of this organelle should totally be repaired during treatment of visceral leishmaniasis (VL). In this paper it has been shown that resumption of normal peroxisomal function could not be attained when one of the existing drugs sodium antimony gluconate (SAG) was used for chemotherapy against VL. Although Leishmania parasite was found to be completely eliminated from host liver and spleen after SAG treatment, normal activities of peroxisomal catalase and superoxide dismutase could not be restored. Also unusual peptides were found to be present due to abnormal proteolytic cleavage of proteins. It is proposed that peroxisomal disorder which exists even after successful chemotherapy of VL may be figured out as one of the possible reasons to develop PKDL. It may also be pointed out that continued effect of peroxisomal disorder even after complete treatment of this parasitic disease may also lead to genetic disorders not yet been explored in post-kala-azar patients.