Human Peroxisomal Disorders Research Articles

TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import.

Most proteins destined for the peroxisomal matrix depend on the peroxisomal targeting signals (PTSs), which require the PTS receptor PEX5, whose deficiency causes fatal human peroxisomal biogenesis disorders (PBDs). TRIM37 gene mutations cause muscle-liver-brain-eye (mulibrey) nanism. We found that TRIM37 localizes in peroxisomal membranes and ubiquitylates PEX5 at K464 by interacting with its C-terminal 51 amino acids (CT51), which is required for PTS protein import. PEX5 mutations (K464A or ΔCT51), or TRIM37 depletion or mutation, reduce PEX5 abundance by promoting its proteasomal degradation, thereby impairing its functions in cargo binding and PTS protein import in human cells. TRIM37 or PEX5 depletion induces apoptosis and enhances sensitivity to oxidative stress, underscoring the cellular requirement for functional peroxisomes. Therefore, TRIM37-mediated ubiquitylation stabilizes PEX5 and promotes peroxisomal matrix protein import, suggesting that mulibrey nanism is a new PBD.

Energy Charge as an Indicator of Pexophagy in Pichia pastoris.

Pichia pastoris is a good model for pexophagy research owing to its diverse pexophagy modes (macropexophagy and micropexophagy) exhibited during carbon-source shift from methanol to other carbon sources. The critical condition that triggers activation of macropexophagy and micropexophagy is important for clarifying the P. pastoris pexophagy mechanism and human peroxisomal disorders. In this study, the pexophagy modes of P. pastoris were confirmed by green fluorescent protein expression and alcohol oxidase and formate dehydrogenase activities. Furthermore, intracellular energy charge (EC) was found to be a determinant of pexophagy activation. During methanol induction, the EC was about 0.5. And the final EC value was related to the pexophagy mode when carbon source switched from methanol to others. Macropexophagy and micropexophagy occurred when the EC increased to 0.6–0.75 and above 0.75, respectively. Thus, different EC values were considered as the important factor to trigger different pexophagy modes in P. pastoris. The results obtained in this study could help in achieving better control of the pexophagy modes to study the pexophagy mechanism.

Human disorders of peroxisome metabolism and biogenesis

Peroxisomes are dynamic organelles that play an essential role in a variety of cellular catabolic and anabolic metabolic pathways, including fatty acid alpha- and beta-oxidation, and plasmalogen and bile acid synthesis. Defects in genes encoding peroxisomal proteins can result in a large variety of peroxisomal disorders either affecting specific metabolic pathways, i.e., the single peroxisomal enzyme deficiencies, or causing a generalized defect in function and assembly of peroxisomes, i.e., peroxisome biogenesis disorders. In this review, we discuss the clinical, biochemical, and genetic aspects of all human peroxisomal disorders currently known. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.

Brain peroxisomes

Peroxisomes are essential organelles in higher eukaryotes as they play a major role in numerous metabolic pathways and redox homeostasis. Some peroxisomal abnormalities, which are often not compatible with life or normal development, were identified in severe demyelinating and neurodegenerative brain diseases. The metabolic roles of peroxisomes, especially in the brain, are described and human brain peroxisomal disorders resulting from a peroxisome biogenesis or a single peroxisomal enzyme defect are listed. The brain abnormalities encountered in these disorders (demyelination, oxidative stress, inflammation, cell death, neuronal migration, differentiation) are described and their pathogenesis are discussed. Finally, the contribution of peroxisomal dysfunctions to the alterations of brain functions during aging and to the development of Alzheimer's disease is considered.

Myelin peroxisomes – Essential organelles for the maintenance of white matter in the nervous system

Peroxisomes are cellular compartments primarily associated with lipid metabolism. Most cell types, including nervous system cells, harbor several hundred of these organelles. The importance of peroxisomes for central nervous system white matter is evidenced by a variety of human peroxisomal disorders with neurological impairment frequently involving the white matter. Moreover, the most frequent childhood white matter disease, X-linked adrenoleukodystrophy, is a peroxisomal disorder. During the past decade advances in imaging techniques have enabled the identification of peroxisomes within the myelin sheath, especially close to nodes of Ranvier. Although the function of myelin peroxisomes is not solved yet on molecular level, recently acquired knowledge suggests a central role for these organelles in axo-glial metabolism. This review focuses on the biology of myelin peroxisomes as well as on the pathology of myelin and myelinated axons that is observed as a consequence of partial or complete peroxisomal dysfunction in the brain.

Biochemical Diagnosis of Peroxisomal Disorders by GC / MS : Egyptian Patients with X-linked Adrenoleukodystrophy

Background: Peroxisomes are organelles responsible mainly for metabolism of lipids and peroxides. Lack of peroxisomes or dysfunction in any of their normal functions is the cellular basis for human peroxisomal disorders (PDs). Aim of the Work: diagnosis of peroxisomal disorders among a high risk group of Egyptian patients using gas chromatography mass spectrometry. Subjects and Methods: Forty six patients suspected to have peroxisomal disorders were included in this study. Their ages ranged from 2 to 20 years. They were referred to The Biochemical Genetics Department, National Research Centre from all over Egypt. Forty one (89%) were males while five were females (11%). Parental consanguinity was positive in 28 cases (61% out of 46). Very long chain fatty acids were quantified after extraction from plasma of all cases using gas chromatography/mass spectrometry (GC/MS) technique. Results: The present study included 46 cases suspected clinically to have one of the peroxisomal disorders; four of them (8.7%) proved to have X-linked adrenoleukodystrophy by quantitative determination of the very long chain fatty acids after extraction from their plasma. The other 42 cases showed normal profile for very long chain fatty acids. Conclusion: This study showed that GC/MS analysis for VLCFA discriminates patients from controls, representing a non-invasive, reliable, specific and sensitive method for the diagnosis of peroxisomal disorders.

Improved analysis of C26:0-lysophosphatidylcholine in dried-blood spots via negative ion mode HPLC-ESI-MS/MS for X-linked adrenoleukodystrophy newborn screening

BackgroundX-linked adrenoleukodystrophy (X-ALD) is the most common human peroxisomal disorder, and is caused by mutations in the peroxisomal transmembrane ALD protein (ALDP, ABCD1). The biochemical defect associated with X-ALD is an accumulation of very long-chain fatty acids (VLCFA, e.g. C24:0 and C26:0), which has been shown to result in the accumulation of C26:0-lysophosphatidylcholine (C26:0-LPC). MethodsWe describe the analysis of C26:0-LPC in dried-blood spots (DBS) using a rapid (30min) and simple extraction procedure, isocratic HPLC resolution of LPC, and structure-specific analysis via negative ion mode tandem mass spectrometry. ResultsIn putative normal DBS specimens from newborns (N=223) C26:0-LPC was 0.09±0.03μmol/l whole blood, while in peroxisomal biogenesis disorder (including X-ALD) patients (N=28) C26:0-LPC was 1.13±0.67μmol/l whole blood. Both multiple reaction monitoring and a neutral loss scan (225.1Da) analysis of DBS were used to analyze LPC. ConclusionsCompared to a previous report of C26:0-LPC analysis in DBS, the method described here is simpler, faster, and more structure-specific for LPC with C26:0 acyl chains.

Plasmalogens participate in very-long-chain fatty acid-induced pathology

Peroxisomes are organelles responsible for multiple metabolic pathways including, the biosynthesis of plasmalogens, a class of phospholipids, and the beta-oxidation of very-long-chain fatty acids (VLCFA). Lack of peroxisomes or dysfunction in any of their normal functions is the cellular basis for human peroxisomal disorders. Here we used mouse models to understand and define the biochemical and cellular determinants that mediate the pathophysiological consequences caused by peroxisomal dysfunctions. We investigated the role and effects of cellular plasmalogens and VLCFA accumulation in liver, testis and nervous tissue using Pex7 and Abcd1 knockout (KO) mice. In addition, we also generated a Pex7:Abcd1 double KO mouse to investigate how different peroxisomal dysfunctions modulate cellular function and pathology. We found that plasmalogens function as fundamental structural phospholipids and protect cells from damage caused by VLCFA accumulation. In testis, plasmalogens protect spermatocytes from VLCFA-induced degeneration and apoptosis. In nervous tissue, we found that gliosis, inflammatory demyelination and axonopathy caused by accumulation of VLCFA are modulated by plasmalogens. Our findings demonstrate the importance of normal peroxisomal functioning and allow the understanding of the pathological causality of peroxisomal dysfunctions. Nervous tissue deficient in plasmalogens is more prone to damage, illustrating the importance of plasmalogens in peroxisomal disorders including Zellweger syndrome and X-linked adrenoleukodystrophy.

Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes.

Very long chain fatty alcohols obtained from plant waxes and beeswax have been reported to lower plasma cholesterol in humans. This review discusses nutritional or regulatory effects produced by wax esters or aliphatic acids and alcohols found in unrefined cereal grains, beeswax, and many plant-derived foods. Reports suggest that 5-20 mg per day of mixed C24-C34 alcohols, including octacosanol and triacontanol, lower low-density lipoprotein (LDL) cholesterol by 21%-29% and raise high-density lipoprotein cholesterol by 8%-15%. Wax esters are hydrolyzed by a bile salt-dependent pancreatic carboxyl esterase, releasing long chain alcohols and fatty acids that are absorbed in the gastrointestinal tract. Studies of fatty alcohol metabolism in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly inter-converted in a fatty alcohol cycle. The metabolism of these compounds is impaired in several inherited human peroxisomal disorders, including adrenoleukodystrophy and Sjögren-Larsson syndrome. Reports on dietary management of these diseases confirm that very long chain fatty acids (VLCFA) are normal constituents of the human diet and are synthesized endogenously. Concentrations of VLCFA in blood plasma increase during fasting and when children are placed on ketogenic diets to suppress seizures. Existing data support the hypothesis that VLCFA exert regulatory roles in cholesterol metabolism in the peroxisome and also alter LDL uptake and metabolism.

RNA interference of peroxisome-related genes in C. elegans: a new model for human peroxisomal disorders.

RNA-mediated interference (RNAi) for the posttranscriptional silencing of genes was used to evaluate the importance of various peroxisomal enzymes and peroxins for the development of Caenorhabditis elegans and to compare the roles of these proteins in the nematode to their roles in yeasts and humans. The nematode counterparts of the human ATP-binding cassette half-transporters, the enzymes alkyldihydroxyacetonephosphate synthase and Delta(3,5)-Delta (2,4)-dienoyl-CoA isomerase, the receptors for peroxisomal membrane and matrix proteins (Pex19p and Pex5p), and components of the docking and translocation machineries for matrix proteins (Pex13p and Pex12p) are essential for the development of C. elegans. Unexpectedly, RNAi silencing of the acyl-CoA synthetase-mediated activation of fatty acids, the alpha- and beta-oxidation of fatty acids, the intraperoxisomal decomposition of hydrogen peroxide, and the peroxins Pex1p, Pex2p, and Pex6p had no apparent effect on C. elegans development. The described analysis of functional gene knockouts through RNAi provides a basis for the use of C. elegans as a valuable model system with which to study the molecular and physiological defects underlying the human peroxisomal disorders.

Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry.

The identification of peroxisomal membrane proteins is very important to understand the import mechanisms of substrates and proteins into these organelles and the pathogenesis of human peroxisomal disorders like the Zellweger Syndrom. Peroxisomal membrane proteins were identified after separation by gel electrophoresis, tryptic digestion and mass spectrometric analysis. Using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), it was possible to identify 45 proteins of isolated yeast peroxisomal membranes.

The peroxisome deficient PEX2 Zellweger mouse: pathologic and biochemical correlates of lipid dysfunction.

Zellweger syndrome is the prototypic human peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. A murine model for this disorder was previously developed by targeted deletion of the PEX2 peroxisomal gene. By labeling neuronal precursor cells in vivo with a mitotic marker, we can demonstrate a delay in neuronal migration in the cerebral cortex of homozygous PEX2 mutant mice. Postnatal PEX2 Zellweger mice develop severe cerebellar defects with abnormal Purkinje cell development and an altered folial pattern. When the PEX2 mutation is placed on an inbred murine genetic background, there is significant embryonic lethality and widespread neuronal lipidosis throughout the brain. Biochemical analysis of PEX2 mutant mice shows the characteristic accumulation of very long chain fatty acids and deficient plasmalogens in a wide variety of tissues. Docosahexaenoic acid levels (DHA; 22:6n-3) were found to be reduced in the brain of mutant mice but were normal in visceral organs at birth. All tissues examined in postnatal mutant mice had reduced DHA. The combined use of morphologic and biochemical analyses in these mice will be essential to elucidate the pathogenesis of this complex peroxisomal disease.

Inverse expression of peroxisomes and connexin-43 in the granulosa cells of the quail follicle.

Studying the regulation of peroxisome (Px) expression could improve our understanding of human peroxisomal disorders. The granulosa of the largest preovulatory quail follicles proved to be a relevant model because (a) Px expression changes according to the follicular maturation stage and (b) Px expression varies regionally according to the distance of the granulosa relative to the germinal disc region containing the female gamete (oocyte). The question was asked whether Px expression is related to the extent of metabolic cell coupling and whether zonal Px variation is causally related to oocytal factors. This was evaluated by the presence of catalase and Cx-43 (marker proteins for peroxisomes and gap junctions, respectively) and by in vitro experiments with granulosa explants. The data obtained show that the expression of Cx-43 and Px is inversely correlated both temporally and spatially. Uncoupling of gap junctions results in an upregulation of alpha-catalase immunofluorescence. This is in agreement with reports that gap junctions are often negatively affected by Px proliferators. The zonal gradient in Px expression appears to be imposed by the oocyte, as is the case for steroidogenesis and proliferative capacity in the granulosa epithelium. (J Histochem Cytochem 48:167-177, 2000)

Predicting the Function and Subcellular Location of Caenorhabditis elegans Proteins Similar to Saccharomyces cerevisiae β-Oxidation Enzymes

The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated. We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g. Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function. Here we have identified C. elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders. PEX-5 interacted strongly in a two-hybrid assay with Gal4p–SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants. A list of C. elegans proteins with similarities to well-characterized yeast β-oxidation enzymes was compiled by homology probing. The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals. Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location. It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes. C. elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a β-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes.

Molecular genetics of peroxisomal disorders.

Twenty five human peroxisomal disorders have been defined at this time. They are subdivided into two major categories: 1) the disorders of peroxisome biogenesis, in which the organelle fails to form normally, and there are defects that involve multiple peroxisomal functions; and 2) disorders that affect single peroxisomal enzymes. During the last five years the molecular defects have been identified in nearly all. These recent advances have several important implications. They have facilitated diagnosis of affected patients. The improved capacity to provide prenatal diagnosis and heterozygote identification has been of great value for genetic counseling and disease prevention. Study of genotype-phenotype correlations has led to a new and more rational classification system. The identification of the molecular defects and the development of animal models have increased understanding of pathogenetic mechanisms, and have led to novel therapeutic approaches.

Stability of alkyl-dihydroxyacetonephosphate synthase in human control and peroxisomal disorder fibroblasts.

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme that plays a key role in ether phospholipid biosynthesis. To determine the turnover of alkyl-DHAP synthase in several peroxisomal disorders, pulse-chase experiments were performed. In control fibroblasts, mature alkyl-DHAP synthase displayed a half-life of 23 +/- 12 h. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, in which alkyl-DHAP synthase cannot be imported into peroxisomes, the enzyme was mainly detected in its precursor form. This precursor form showed a much shorter half-life, 5 +/- 2 h. In contrast, when the precursor protein accumulated inside the peroxisome of a particular neonatal adrenoleukodystrophy cell line in which processing does not take place, a half-life of 18 +/- 8 h, resembling that of the mature protein in controls, was observed. In a cell line from a patient with a single deficiency in the activity of alkyl-DHAP synthase, the mature form was detected and its radioactivity decreased with a half-life of 16 +/- 7 h. Collectively, these results provide an explanation for the instability of alkyl-DHAP synthase outside its target organelle. Additionally, they indicate that both the precursor and mature form of alkyl-DHAP synthase exhibit considerable intraperoxisomal turnover.

Stability of Alkyl-Dihydroxyacetonephosphate Synthase in Human Control and Peroxisomal Disorder Fibroblasts

Stability of Alkyl-Dihydroxyacetonephosphate Synthase in Human Control and Peroxisomal Disorder Fibroblasts

Genotype–Phenotype Correlations in Disorders of Peroxisome Biogenesis

Genetically determined human peroxisomal disorders are subdivided into two major categories: disorders of peroxisome biogenesis (PBD), in which the organelle is not formed normally, and those that involve a single peroxisomal enzyme. Twelve PBD have been identified, and the molecular defects have been defined in 10. All involve defects in the import of proteins into the organelle. Factors required for this import are now referred to as peroxins (PEX) and form the basis of a new and preferred classification system. The PBD are associated with four clinical phenotypes, named before their association with the organelle was recognized: Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD), and rhizomelic chondrodysplasia punctata (RCDP). The first three are associated with 9 of the 10 PEX defects that have been defined so far, and represent a clinical continuum with variant severity, with ZS the most severe, NALD intermediate, and IRD the least severe. RCDP is associated with PEX7. Genotype–phenotype correlations are complicated by the fact that the clinical manifestations of the ZS–NALD–IRD continuum can be mimicked by disorders that affect single enzymes of peroxisomal fatty acid oxidation, and PEX7 by disorders of plasmalogen synthesis enzymes. Furthermore, clinical manifestations of each of the PEX disorders may vary. Phenotypic expression varies with the nature of the mutation, the milder phenotypes being associated with mutations that do not abolish function completely, or with mosaicism. Definition of the molecular defects is of great value for genetic counseling and may be of aid in establishing prognosis.

Immunological analyses of alkyl-dihydroxyacetonephosphate synthase in human peroxisomal disorders

Alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) is a peroxisomal enzyme involved in the biosynthesis of ether phospholipids. To localize the enzyme in human peroxisomal disorders, indirect immunofluorescence and immunoblot analysis was performed. In Zellweger syndrome and rhizomelic chondrodysplasia punctata fibroblast cell lines, alkyl-DHAP synthase protein levels on immunoblots were strongly decreased and residual immunofluorescence was diffusely localized throughout the cytoplasm. In a particular neonatal adrenoleukodystrophy cell line, characterized by the absence of a functional peroxisomal targeting signal 1 receptor, the precursor form of the enzyme was detected in Western blots at levels comparable to that of the mature enzyme in control fibroblasts. Similarly, fibroblasts from patients with a single deficiency in the activity of either alkyl-DHAP synthase or DHAP-acyltransferase showed normal levels of the mature alkyl-DHAP synthase protein on immunoblots. Immunofluorescence experiments revealed a peroxisomal localization of both the precursor and the mature form of the enzyme. Collectively, these results visualize the peroxisomal localization of alkyl-DHAP synthase, indicate that the enzyme is unstable outside its target organelle and explain that normal enzyme protein levels found in some peroxisomal disorders result from protection against cytoplasmic degradation through import into peroxisomes. Additionally, alkyl-DHAP synthase could be detected in rat mesangial cells and murine NIH-3R3 fibroblasts by immunofluorescence as well as immunoblot analysis. Immunoelectron microscopy showed that the enzyme is predominantly located on the lumenal side of the peroxisomal membrane in rat and guinea pig liver.

Molecular Genetics of Peroxisomal Disorders

Twenty five human peroxisomal disorders have been defined at this time. They are subdivided into two major categories: 1) the disorders of peroxisome biogenesis, in which the organelle fails to form normally, and there are defects that involve multiple peroxisomal functions; and 2) disorders that affect single peroxisomal enzymes. During the last five years the molecular defects have been identified in nearly all. These recent advances have several important implications. They have facilitated diagnosis of affected patients. The improved capacity to provide prenatal diagnosis and heterozygote identification has been of great value for genetic counseling and disease prevention. Study of genotype-phenotype correlations has led to a new and more rational classification system. The identification of the molecular defects and the development of animal models have increased understanding of pathogenetic mechanisms, and have led to novel therapeutic approaches.