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1.  Drosophila Carrying Pex3 or Pex16 Mutations Are Models of Zellweger Syndrome That Reflect Its Symptoms Associated with the Absence of Peroxisomes 
PLoS ONE  2011;6(8):e22984.
The peroxisome biogenesis disorders (PBDs) are currently difficult-to-treat multiple-organ dysfunction disorders that result from the defective biogenesis of peroxisomes. Genes encoding Peroxins, which are required for peroxisome biogenesis or functions, are known causative genes of PBDs. The human peroxin genes PEX3 or PEX16 are required for peroxisomal membrane protein targeting, and their mutations cause Zellweger syndrome, a class of PBDs. Lack of understanding about the pathogenesis of Zellweger syndrome has hindered the development of effective treatments. Here, we developed potential Drosophila models for Zellweger syndrome, in which the Drosophila pex3 or pex16 gene was disrupted. As found in Zellweger syndrome patients, peroxisomes were not observed in the homozygous Drosophila pex3 mutant, which was larval lethal. However, the pex16 homozygote lacking its maternal contribution was viable and still maintained a small number of peroxisome-like granules, even though PEX16 is essential for the biosynthesis of peroxisomes in humans. These results suggest that the requirements for pex3 and pex16 in peroxisome biosynthesis in Drosophila are different, and the role of PEX16 orthologs may have diverged between mammals and Drosophila. The phenotypes of our Zellweger syndrome model flies, such as larval lethality in pex3, and reduced size, shortened longevity, locomotion defects, and abnormal lipid metabolisms in pex16, were reminiscent of symptoms of this disorder, although the Drosophila pex16 mutant does not recapitulate the infant death of Zellweger syndrome. Furthermore, pex16 mutants showed male-specific sterility that resulted from the arrest of spermatocyte maturation. pex16 expressed in somatic cyst cells but not germline cells had an essential role in the maturation of male germline cells, suggesting that peroxisome-dependent signals in somatic cyst cells could contribute to the progression of male germ-cell maturation. These potential Drosophila models for Zellweger syndrome should contribute to our understanding of its pathology.
PMCID: PMC3149631  PMID: 21826223
2.  Identification of Novel Mutations and Sequence Variation in the Zellweger Syndrome Spectrum of Peroxisome Biogenesis Disorders 
Human mutation  2009;30(3):E467-E480.
Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive neurodegenerative disorders that affect multiple organ systems. Approximately 80% of PBD patients are classified in the Zellweger syndrome spectrum (PBD-ZSS). Mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes are found in approximately 90% of PBD-ZSS patients. Here, we sequenced the coding regions and splice junctions of these five genes in 58 PBD-ZSS cases previously subjected to targeted sequencing of a limited number of PEX gene exons. In our cohort, 71 unique sequence variants were identified, including 18 novel mutations predicted to disrupt protein function and 2 novel silent variants. We identified 4 patients who had two deleterious mutations in one PEX gene and a third deleterious mutation in a second PEX gene. For two such patients, we conducted cell fusion complementation analyses to identify the defective gene responsible for aberrant peroxisome assembly. Overall, we provide empirical data to estimate the relative fraction of disease-causing alleles that occur in the coding and splice junction sequences of these five PEX genes and the frequency of cases where mutations occur in multiple PEX genes. This information is beneficial for efforts aimed at establishing rapid and sensitive clinical diagnostics for PBD-ZSS patients and interpreting the results from these genetic tests.
PMCID: PMC2649967  PMID: 19105186
peroxisome biogenesis disorders; Zellweger syndrome; PBD-ZSS; neonatal adrenoleukodystrophy; infantile Refsum disease; PEX1; PEX6; PEX10; PEX12
3.  Pex12 Interacts with Pex5 and Pex10 and Acts Downstream of Receptor Docking in Peroxisomal Matrix Protein Import 
The Journal of Cell Biology  1999;147(4):761-774.
Peroxisomal matrix protein import requires PEX12, an integral peroxisomal membrane protein with a zinc ring domain at its carboxy terminus. Mutations in human PEX12 result in Zellweger syndrome, a lethal neurological disorder, and implicate the zinc ring domain in PEX12 function. Using two-hybrid studies, blot overlay assays, and coimmunoprecipitation experiments, we observed that the zinc-binding domain of PEX12 binds both PEX5, the PTS1 receptor, and PEX10, another integral peroxisomal membrane protein required for peroxisomal matrix protein import. Furthermore, we identified a patient with a missense mutation in the PEX12 zinc-binding domain, S320F, and observed that this mutation reduces the binding of PEX12 to PEX5 and PEX10. Overexpression of either PEX5 or PEX10 can suppress this PEX12 mutation, providing genetic evidence that these interactions are biologically relevant. PEX5 is a predominantly cytoplasmic protein and previous PEX5-binding proteins have been implicated in docking PEX5 to the peroxisome surface. However, we find that loss of PEX12 or PEX10 does not reduce the association of PEX5 with peroxisomes, demonstrating that these peroxins are not required for receptor docking. These and other results lead us to propose that PEX12 and PEX10 play direct roles in peroxisomal matrix protein import downstream of the receptor docking event.
PMCID: PMC2156163  PMID: 10562279
PTS1 receptor; PEX5; PEX10; Zellweger syndrome; peroxisome biogenesis disorder
4.  Peroxisome Synthesis in the Absence of Preexisting Peroxisomes  
The Journal of Cell Biology  1999;144(2):255-266.
Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2–3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.
PMCID: PMC2132891  PMID: 9922452
membrane biogenesis; Zellweger syndrome; peroxisomal membrane protein import; PEX16; peroxisome biogenesis disorders
5.  Rational diagnostic strategy for Zellweger syndrome spectrum patients 
Zellweger syndrome spectrum (ZSS) comprises a clinically and genetically heterogeneous disease entity, which is caused by mutations in any of the 12 different human PEX genes leading to impaired biogenesis of the peroxisome. Patients potentially suffering from ZSS are diagnosed biochemically by measuring elevated levels of very long chain fatty acids, pristanic acid and phytanic acid in plasma and serum and reduced levels of ether phospholipids in erythrocytes. Published reports on diagnostic procedures for ZSS patients are restricted either to biochemical markers or to defined mutations in a subset of PEX genes. Clarification of the primary genetic defect in an affected patient is crucial for genetic counselling, carrier testing or prenatal diagnosis. In this study, we present a rational diagnostic strategy for patients suspected of ZSS. By combining cell biology and molecular genetic methods in an appropriate sequence, we were able to detect the underlying mutation in various PEX genes within adequate time and cost. We applied this method on 90 patients who presented at our institute, Department of Pediatrics and Pediatric Neurology at Georg August University, and detected 174 mutant alleles within six different PEX genes, including two novel deletions and three new missense mutations in PEX6. Furthermore, this strategy will extend our knowledge on genotype–phenotype correlation in various PEX genes. It will contribute to a better understanding of ZSS pathogenesis, allowing the investigation of the effects of diverse mutations on the interaction between PEX proteins and peroxisomal function in vivo.
PMCID: PMC2947092  PMID: 19142205
Zellweger syndrome spectrum; peroxisome; diagnosis; PEX mutation
6.  A Drosophila model for the Zellweger spectrum of peroxisome biogenesis disorders 
Disease Models & Mechanisms  2011;4(5):659-672.
Human peroxisome biogenesis disorders are lethal genetic diseases in which abnormal peroxisome assembly compromises overall peroxisome and cellular function. Peroxisomes are ubiquitous membrane-bound organelles involved in several important biochemical processes, notably lipid metabolism and the use of reactive oxygen species for detoxification. Using cultured cells, we systematically characterized the peroxisome assembly phenotypes associated with dsRNA-mediated knockdown of 14 predicted Drosophila homologs of PEX genes (encoding peroxins; required for peroxisome assembly and linked to peroxisome biogenesis disorders), and confirmed that at least 13 of them are required for normal peroxisome assembly. We also demonstrate the relevance of Drosophila as a genetic model for the early developmental defects associated with the human peroxisome biogenesis disorders. Mutation of the PEX1 gene is the most common cause of peroxisome biogenesis disorders and is one of the causes of the most severe form of the disease, Zellweger syndrome. Inherited mutations in Drosophila Pex1 correlate with reproducible defects during early development. Notably, Pex1 mutant larvae exhibit abnormalities that are analogous to those exhibited by Zellweger syndrome patients, including developmental delay, poor feeding, severe structural abnormalities in the peripheral and central nervous systems, and early death. Finally, microarray analysis defined several clusters of genes whose expression varied significantly between wild-type and mutant larvae, implicating peroxisomal function in neuronal development, innate immunity, lipid and protein metabolism, gamete formation, and meiosis.
PMCID: PMC3180231  PMID: 21669930
7.  Characterization of two common 5' polymorphisms in PEX1 and correlation to survival in PEX1 peroxisome biogenesis disorder patients 
BMC Medical Genetics  2011;12:109.
Mutations in PEX1 are the most common primary cause of Zellweger syndrome. In addition to exonic mutations, deletions and splice site mutations two 5' polymorphisms at c.-137 and c.-53 with a potential influence on PEX1 protein levels have been described in the 5' untranslated region (UTR) of the PEX1 gene.
We used RACE and in silico promoter prediction analysis to study the 5' UTR of PEX1. We determined the distribution of PEX1 5' polymorphisms in a cohort of 30 Zellweger syndrome patients by standard DNA sequencing. 5' polymorphisms were analysed in relation to the two most common mutations in PEX1 and were incorporated into a novel genotype-phenotype analysis by correlation of three classes of PEX1 mutations with patient survival.
We provide evidence that the polymorphism 137 bp upstream of the ATG codon is not part of the UTR, rendering it a promoter polymorphism. We show that the first, but not the second most common PEX1 mutation arose independently of a specific upstream polymorphic constellation. By genotype-phenotype analysis we identified patients with identical exonic mutation and identical 5' polymorphisms, but strongly differing survival.
Our study suggests that two different types of PEX1 5' polymorphisms have to be distinguished: a 5' UTR polymorphism at position c.-53 and a promoter polymorphism 137 bp upstream of the PEX1 start codon. Our results indicate that the exonic PEX1 mutation correlates with patient survival, but the two 5' polymorphisms analysed in this study do not have to be considered for diagnostic and/or prognostic purposes.
PMCID: PMC3167756  PMID: 21846392
8.  Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype 
Molecular and Cellular Biology  2003;23(16):5947-5957.
Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13−/− mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.
PMCID: PMC166343  PMID: 12897163
9.  PEX12, the Pathogenic Gene of Group III Zellweger Syndrome: cDNA Cloning by Functional Complementation on a CHO Cell Mutant, Patient Analysis, and Characterization of Pex12p 
Molecular and Cellular Biology  1998;18(7):4324-4336.
Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11–20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.
PMCID: PMC109016  PMID: 9632816
10.  Inhibitors of Copi and Copii Do Not Block PEX3-Mediated Peroxisome Synthesis 
The Journal of Cell Biology  2000;149(7):1345-1360.
In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.
PMCID: PMC2175136  PMID: 10871277
Zellweger syndrome; membrane biogenesis; protein import; vesicle traffic; peroxisome biogenesis disorders
11.  Saccharomyces cerevisiae PTS1 Receptor Pex5p Interacts with the SH3 Domain of the Peroxisomal Membrane Protein Pex13p in an Unconventional, Non-PXXP–related Manner 
Molecular Biology of the Cell  2000;11(11):3963-3976.
A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W204XXQF208, that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Δ cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.
PMCID: PMC15050  PMID: 11071920
12.  PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress 
Disease Models & Mechanisms  2010;4(1):104-119.
Delayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.
PMCID: PMC3014351  PMID: 20959636
13.  Genetic and clinical aspects of Zellweger spectrum patients with PEX1 mutations 
Journal of Medical Genetics  2005;42(9):e58.
Objective: To analyse the PEX1 gene, the most common cause for peroxisome biogenesis disorders (PBD), in a consecutive series of patients with Zellweger spectrum.
Methods: Mutations were detected by different methods including SSCP analyses as a screening technique on the basis of genomic or cDNA, followed by direct sequencing of PCR fragments with an abnormal electrophoresis pattern.
Results: 33 patients were studied. Two common mutations, c.2528G→A, G843D and c.2098_2098insT, I700YfsX42, accounted for over 80% of all abnormal PEX1 alleles, emphasising their diagnostic relevance. Most PEX1 mutations were distributed over the two AAA cassettes with the two functional protein domains, D1 and D2, and the highly conserved Walker motifs. Phenotypic severity of Zellweger spectrum in CG1 depended on the effect of the mutation on the PEX1 protein, peroxin 1. PEX1 mutations could be divided into two classes of genotype–phenotype correlation: class I mutations led to residual PEX1 protein levels and function and a milder phenotype; class II mutations almost abolished PEX1 protein levels and function, resulting in a severe phenotype. Compound heterozygote patients for a class I and class II mutation had an intermediate phenotype.
Conclusions: Molecular confirmation of the clinical and biochemical diagnosis will allow the prediction of the clinical course of disease in individual PBD cases.
PMCID: PMC1736134  PMID: 16141001
14.  Arginine improves peroxisome functioning in cells from patients with a mild peroxisome biogenesis disorder 
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.
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.
Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX1, 6 and 12 can be improved by arginine.
Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisome biogenesis disorder.
PMCID: PMC3844471  PMID: 24016303
Peroxisome biogenesis disorder; Zellweger spectrum disorder; Misfolded protein; Peroxisomal mosaicism; Arginine; Therapy
15.  PEX11β Deficiency Is Lethal and Impairs Neuronal Migration but Does Not Abrogate Peroxisome Function 
Molecular and Cellular Biology  2002;22(12):4358-4365.
Zellweger syndrome is a lethal neurological disorder characterized by severe defects in peroxisomal protein import. The resulting defects in peroxisome metabolism and the accumulation of peroxisomal substrates are thought to cause the other Zellweger syndrome phenotypes, including neuronal migration defects, hypotonia, a developmental delay, and neonatal lethality. These phenotypes are also manifested in mouse models of Zellweger syndrome generated by disruption of the PEX5 or PEX2 gene. Here we show that mice lacking peroxisomal membrane protein PEX11β display several pathologic features shared by these mouse models of Zellweger syndrome, including neuronal migration defects, enhanced neuronal apoptosis, a developmental delay, hypotonia, and neonatal lethality. However, PEX11β deficiency differs significantly from Zellweger syndrome and Zellweger syndrome mice in that it is not characterized by a detectable defect in peroxisomal protein import and displays only mild defects in peroxisomal fatty acid β-oxidation and peroxisomal ether lipid biosynthesis. These results demonstrate that the neurological pathologic features of Zellweger syndrome can occur without peroxisomal enzyme mislocalization and challenge current models of Zellweger syndrome pathogenesis.
PMCID: PMC133847  PMID: 12024045
16.  Defective PEX gene products correlate with the protein import, biochemical abnormalities, and phenotypic heterogeneity in peroxisome biogenesis disorders 
Journal of Medical Genetics  1999;36(10):779-781.
Peroxisome biogenesis disorders (PBD) comprise three phenotypes including Zellweger syndrome (ZS) (the most severe), neonatal adrenoleucodystrophy, and infantile Refsum disease (IRD) (the most mild), and can be classified into at least 12 genetic complementation groups, which are not predictive of the phenotypes. Several pathogenic genes for PBD groups have been identified, but the relationship between the defective gene products and phenotypic heterogeneity has remained unclear. We identified a mutation in the PEX2 gene in an IRD patient with compound heterozygosity for a missense mutation and the known nonsense mutation detected in ZS patients. In transfection experiments using the peroxisome deficient CHO mutant, Z65 with a nonsense mutation in the PEX2 gene, we noted the E55K mutation had mosaic activities of peroxisomal protein import machinery and residual activities of peroxisomal functions, including dihydroxyacetone phosphate acyltransferase and β oxidation of very long chain fatty acids. The nonsense mutation severely affects these peroxisomal functions as well as the protein import. These data suggest that allelic heterogeneity of the PEX gene affects the peroxisomal protein import and functions and regulates the clinical severity in PBD.

Keywords: Zellweger syndrome; infantile Refsum disease; PEX gene; mosaic
PMCID: PMC1734244  PMID: 10528859
17.  Disturbed Cholesterol Homeostasis in a Peroxisome-Deficient PEX2 Knockout Mouse Model 
We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2−/− mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2−/− mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2−/− mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2−/− mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2−/− mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2−/− mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2−/− mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2−/− mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.
PMCID: PMC303355  PMID: 14673138
18.  Identification of a novel PEX14 mutation in Zellweger syndrome 
BMJ Case Reports  2009;2009:bcr07.2008.0503.
Here we report a patient with Zellweger syndrome, who presented at the age of 3 months with icterus, dystrophy, axial hypotonia, and hepatomegaly. Abnormal findings of metabolic screening tests included hyperbilirubinaemia, hypoketotic dicarboxylic aciduria, increased C26:0 and decreased C22:0 plasma levels, and strongly reduced plasmalogen concentrations. In fibroblasts, both peroxisomal α- and β-oxidation were impaired. Liver histology revealed bile duct paucity, cholestasis, arterial hyperplasia, very small branches of the vena portae, and parenchymatic destruction. Immunocytochemical analysis of cultured fibroblasts demonstrated that the cells contain peroxisomal remnants lacking apparent matrix protein content and PEX14, a central membrane component of the peroxisomal matrix protein import machinery. Transfection of fibroblasts with a plasmid coding for wild-type PEX14 restored peroxisomal matrix protein import. Mutational analysis of this gene revealed a genomic deletion leading to the deletion of exon 3 from the coding DNA (c.85-?_170+?del) and a concomitant change of the reading frame (p.[Ile29_Lys56del;Gly57GlyfsX2]).
PMCID: PMC3027610  PMID: 21686775
19.  Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import 
Molecular and Cellular Biology  2002;22(6):1639-1655.
Two isoforms of the peroxisomal targeting signal type 1 (PTS1) receptor, termed Pex5pS and (37-amino-acid-longer) Pex5pL, are expressed in mammals. Pex5pL transports PTS1 proteins and Pex7p-PTS2 cargo complexes to the initial Pex5p-docking site, Pex14p, on peroxisome membranes, while Pex5pS translocates only PTS1 cargoes. Here we report functional Pex5p domains responsible for interaction with peroxins Pex7p, Pex13p, and Pex14p. An N-terminal half, such as Pex5pL(1-243), comprising amino acid residues 1 to 243, bound to Pex7p, Pex13p, and Pex14p and was sufficient for restoring the impaired PTS2 import of pex5 cell mutants, while the C-terminal tetratricopeptide repeat motifs were required for PTS1 binding. N-terminal Pex5p possessed multiple Pex14p-binding sites. Alanine-scanning analysis of the highly conserved seven (six in Pex5pS) pentapeptide WXXXF/Y motifs residing at the N-terminal region indicated that these motifs were essential for the interaction of Pex5p with Pex14p and Pex13p. Moreover, mutation of several WXXXF/Y motifs did not affect the PTS import-restoring activity of Pex5p, implying that the binding of Pex14p to all of the WXXXF/Y sites was not a prerequisite for the translocation of Pex5p-cargo complexes. Pex5p bound to Pex13p at the N-terminal part, not to the C-terminal SH3 region, via WXXXF/Y motifs 2 to 4. PTS1 and PTS2 import required the interaction of Pex5p with Pex14p but not with Pex13p, while Pex5p binding to Pex13p was essential for import of catalase with PTS1-like signal KANL. Pex5p recruited PTS1 proteins to Pex14p but not to Pex13p. Pex14p and Pex13p formed a complex with PTS1-loaded Pex5p but dissociated in the presence of cargo-unloaded Pex5p, implying that PTS cargoes are released from Pex5p at a step downstream of Pex14p and upstream of Pex13p. Thus, Pex14p and Pex13p very likely form mutually and temporally distinct subcomplexes involved in peroxisomal matrix protein import.
PMCID: PMC135613  PMID: 11865044
20.  The Peroxisome Biogenesis Factors Pex4p, Pex22p, Pex1p, and Pex6p Act in the Terminal Steps of Peroxisomal Matrix Protein Import 
Molecular and Cellular Biology  2000;20(20):7516-7526.
Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Δ mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.
PMCID: PMC86304  PMID: 11003648
21.  Yarrowia lipolytica Cells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes 
Molecular Biology of the Cell  2001;12(11):3353-3364.
PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid–containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane.
PMCID: PMC60260  PMID: 11694572
22.  Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica. 
Molecular and Cellular Biology  1997;17(9):5210-5226.
We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.
PMCID: PMC232372  PMID: 9271399
23.  Pex15p of Saccharomyces cerevisiae Provides a Molecular Basis for Recruitment of the AAA Peroxin Pex6p to Peroxisomal Membranes 
Molecular Biology of the Cell  2003;14(6):2226-2236.
The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.
PMCID: PMC194873  PMID: 12808025
24.  Fine Mapping and Association Studies of a High-Density Lipoprotein Cholesterol Linkage Region on Chromosome 16 in French-Canadian Subjects 
Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for cardiovascular disease. To identify novel genetic variants that contribute to HDL-C, we performed genome-wide scans and quantitative association studies in two study samples: a Quebec-wide study consisting of 11 multi-generational families and a study of 61 families from the Saguenay–Lac St-Jean (SLSJ) region of Quebec. The heritability of HDL-C in these study samples was 0.73 and 0.49 respectively. Variance components linkage methods identified a LOD score of 2.61, at 98 cM near the marker D16S515 in the Quebec-wide families and a LOD score of 2.96 at 86 cM, near the marker D16S2624 in the SLSJ families. In the Quebec-wide sample, four families demonstrated segregation over a 25.5 cM (18 Mb) region, which was further reduced to 6.6 Mb with additional markers. The coding regions of all genes within this region were sequenced. A missense variant in CHST6, segregated in four families and with additional families we observed a p value of 0.015 for this variant. However, an association study of this SNP in unrelated Quebec-wide samples was not significant. We also identified a SNP (rs11646677) in the same region, which was significantly associated with low HDL-C (p=0.016) in the SLSJ study sample. In addition, RT-PCR results from cultured cells demonstrated a significant difference in expression of CHST6 and KIAA1576, another gene in the region. Our data constitute additional evidence for a locus on chromosome 16q23-24 that affects HDL-C levels in two independent French Canadian studies.
PMCID: PMC2824775  PMID: 19844255
high-density lipoprotein cholesterol; family study; complex traits; coronary heart disease; gene identification
25.  Fine mapping and association studies of a high-density lipoprotein cholesterol linkage region on chromosome 16 in French-Canadian subjects 
Low levels of high-density lipoprotein cholesterol (HDL-C) are an independent risk factor for cardiovascular disease. To identify novel genetic variants that contribute to HDL-C, we performed genome-wide scans and quantitative association studies in two study samples: a Quebec-wide study consisting of 11 multigenerational families and a study of 61 families from the Saguenay–Lac St-Jean (SLSJ) region of Quebec. The heritability of HDL-C in these study samples was 0.73 and 0.49, respectively. Variance components linkage methods identified a LOD score of 2.61 at 98 cM near the marker D16S515 in Quebec-wide families and an LOD score of 2.96 at 86 cM near the marker D16S2624 in SLSJ families. In the Quebec-wide sample, four families showed segregation over a 25.5-cM (18 Mb) region, which was further reduced to 6.6 Mb with additional markers. The coding regions of all genes within this region were sequenced. A missense variant in CHST6 segregated in four families and, with additional families, we observed a P value of 0.015 for this variant. However, an association study of this single-nucleotide polymorphism (SNP) in unrelated Quebec-wide samples was not significant. We also identified an SNP (rs11646677) in the same region, which was significantly associated with a low HDL-C (P=0.016) in the SLSJ study sample. In addition, RT-PCR results from cultured cells showed a significant difference in the expression of CHST6 and KIAA1576, another gene in the region. Our data constitute additional evidence for a locus on chromosome 16q23-24 that affects HDL-C levels in two independent French-Canadian studies.
PMCID: PMC2824775  PMID: 19844255
HDL-C; family study; complex traits; coronary heart disease; gene identification

Results 1-25 (509761)