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.     

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.     

Peroxisomal disorders: the single peroxisomal enzyme deficiencies

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

Going against the flow: A case for peroxisomal protein export

Peroxisomes play a crucial role in regulating cellular metabolism, providing compartments where metabolic pathways can be contained and controlled. Their importance is underlined by the developmental brain disorders caused by peroxisome malfunction, while disturbances in peroxisome function also contribute to ageing. As peroxisomes do not contain DNA, they rely on an active transport system to obtain the full quota of proteins required for function. Organelle protein transport however, is rarely a one-way process and exciting recent data have demonstrated that peroxisomes can selectively export membrane and matrix proteins to fulfil specific functions. This review will summarise the current knowledge on peroxisomal membrane and matrix protein export, discussing the mechanisms underlying export as well as the role of peroxisomal protein export in peroxisomal and cellular function.     

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.     

Structural and functional aspects of peroxisomal membranes in yeasts

Abstract: The peroxisomal membrane compartmentalizes specific metabolic functions in the intermediary metabolism of various aerobic eukarya. In yeast, peroxisomal membranes are typified by their small width (±7–8 nm) and absence of large integral membrane proteins in freeze-etch replicas. They show a unique polypeptide profile which, in contrast to their phospholipid composition, differs from that of other membranes in the cell. Part of these proteins are substrate- inducible and are probably related to specific peroxisomal function(s). In vivo, the observed proton motive force across the peroxisomal membrane may play a role in the function of the organelle in that it contributes to the driving force required for selective transport of various enzyme substrates and/or metabolic intermediates. To date only few peroxisomal membrane proteins (PMPs) have been functionally characterized. A major constitutive 31-kDa PMP present in the peroxisomal membrane of Hansenula polymorpha has been purified and was shown to display poreforming properties. In addition, a peroxisomal H⁺-ATPase has been identified which most probably is involved in the generation/maintenance of the in vivo pH gradient across the peroxisomal membrane. Other functions of peroxisomal membrane proteins remain obscure although the first genes encoding yeast PMPs are now being cloned and sequenced. Studies on peroxisome-deficient yeast mutants revealed that specific peroxisome functions are strictly dependent on the intactness of the peroxisomal membrane. In this contribution several examples are presented of metabolic disorders due to peroxisomal malfunction in yeast.     

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.     

Sorting and function of peroxisomal membrane proteins

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.     

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.     

Quantitative proteomic comparison of mouse peroxisomes from liver and kidney

The peroxisome plays a central role in the catabolic and anabolic pathways that contribute to the lipid homeostasis. Besides this main function, this organelle has gained functional diversity. Although several approaches have been used for peroxisomal proteome analysis, a quantitative protein expression analysis of peroxisomes from different tissues has not been elucidated yet. Here, we applied a 2-DE-based method on mouse liver and kidney peroxisomal enriched fractions to study the tissue-dependent protein expression. Ninety-one spots were identified from the 2-DE maps from pH 3.0-10.0 and 51 spots from the basic range corresponding to 31 peroxisomal proteins, 10 putative peroxisomal, 6 cytosolic, 17 mitochondrial and 1 protein from endoplasmic reticulum. Based on the identification and on the equivalent quality of both tissue preparations, the differences emerging from the comparison could be quantified. In liver, proteins involved in pathways such as alpha- and beta-oxidation, isoprenoid biosynthesis, amino acid metabolism and purine and pyrimidine metabolism were more abundant whereas in kidney, proteins from the straight-chain fatty acid beta-oxidation were highly expressed. These results indicate that tissue-specific functional classes of peroxisomal proteins could be relevant to study peroxisomal cellular responses or pathologies. Finally, a web-based peroxisomal proteomic database was built.     

Omega-3 fatty acids and the peroxisome

The interactions between the omega-3 unsaturated fatty acids and peroxisomal function have been reviewed, in order to update and integrate knowledge in this area. Following a brief retrospective of the major clinical involvements of these fatty acids, the participation of the peroxisome in their metabolism has been appraised - the peroxisome being shown to exert a major influence on both the synthesis and degradation of the omega-3 fatty acids, with these effects flowing on to the widespread physiological implications of the derivative eicosanoids. Interactions between the omega-3 and omega-6 families of fatty acids have been discussed, as have the interdependent phenomena of peroxisome proliferation, membrane remodelling and cellular signalling. Amongst the signalling involvements covered were those of steroid hormone receptor superfamily, the phosphatidy1choline cycle, and the regulatory influences of oxygen free radicals. Comment has also been included on the separate biological roles of the individual omega-3 fatty acids, their influence on differential gene function, and on the molecular mechanisms of their pharmacological effects. It is concluded that the peroxisome is intimately involved in directing the metabolism and physiological influence of the omega-3 unsaturated fatty acids, and that this organelle merits much greater emphasis in future research aimed at unravelling the profound biological effects of these unique and multipotent compounds.     

Peroxisome metabolism and cellular aging

The essential role of peroxisomes in fatty acid oxidation, anaplerotic metabolism, and hydrogen peroxide turnover is well established. Recent findings suggest that these and other related biochemical processes governed by the organelle may also play a critical role in regulating cellular aging. The goal of this review is to summarize and integrate into a model the evidence that peroxisome metabolism actually helps define the replicative and chronological age of a eukaryotic cell. In this model, peroxisomal reactive oxygen species (ROS) are seen as altering organelle biogenesis and function, and eliciting changes in the dynamic communication networks that exist between peroxisomes and other cellular compartments. At low levels, peroxisomal ROS activate an anti-aging program in the cell; at concentrations beyond a specific threshold, a pro-aging course is triggered.     

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.     

Peroxisomes start their life in the endoplasmic reticulum

Peroxisomes belong to the ubiquitous organelle repertoire of eukaryotic cells. They contribute to cellular metabolism in various ways depending on species, but a consistent feature is the presence of enzymes to degrade fatty acids. Due to the pioneering work of DeDuve and coworkers, peroxisomes were in the limelight of cell biology in the sixties with a focus on their metabolic role. During the last decade, interest in peroxisomes has been growing again, this time with focus on their origin and maintenance. This has resulted in our understanding how peroxisomal proteins are targeted to the organelle and imported into the organellar matrix or recruited into the single membrane surrounding it. With respect to the formation of peroxisomes, the field is divided. The long-held view formulated in 1985 by Lazarow and Fujiki (Lazarow PB, Fujiki Y. Biogenesis of peroxisomes. Annu Rev Cell Biol 1985; 1: 489–530) is that we are dealing with autonomous organelles multiplying by growth and division. This view is being challenged by various observations that call attention to a more active contribution of the ER to peroxisome formation. Our contribution to this debate consists of recent observations using immuno-electronmicroscopy and electron tomography in mouse dendritic cells that show the peroxisomal membrane to be derived from the ER.     

Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.     

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative 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.     

Peroxisomal Disorders: Clinical,Biochemical, and Molecular Aspects

Peroxisomes are subcellular organelles catalyzing a number of indispensable functions in cellular metabolism. The importance of peroxisomes in man is stressed by the existence of an expanding group of genetic diseases in which there is an impairment in one or more peroxisomal functions. Much has been learned in recent years about these functions and many of the enzymes involved have been characterized, purified and their cDNAs cloned. This has allowed resolution of the enzymatic and molecular basis of many of the single peroxisomal enzyme deficiencies. Similarly, the molecular basis of the peroxisome biogenesis disorders is also being resolved rapidly thanks to the successful use of CHO as well as yeast mutants. In this paper we will provide an overview of the peroxisomal disorders with particular emphasis on their clinical, biochemical and molecular characteristics.     

The evolutionary origin of peroxisomes: an ER-peroxisome connection

The peroxisome is an essential eukaryotic organelle, crucial for lipid metabolism and free radical detoxification, development, differentiation, and morphogenesis from yeasts to humans. Loss of peroxisomes invariably leads to fatal peroxisome biogenesis disorders in man. The evolutionary origin of peroxisomes remains unsolved; proposals for either a symbiogenetic or cellular membrane invagination event are unconclusive. To address this question, we have probed with a peroxisomal proteome, an "ensemble" of 19 representative eukaryotic complete genomes. Molecular phylogenetic and sequence comparison tools allowed us to identify four proteins as peroxisomal markers for unequivocal in silico peroxisome detection. We have then detected the Apicomplexa phylum as the first group of organisms devoid of peroxisomes, in the presence of mitochondria. Finally, we deliver evidence against a prokaryotic ancestor of peroxisomes: (1) the peroxisomal membrane is composed of purely eukaryotic bricks and is thus useful to trace the eukaryotes in their evolutionary paths and (2) the peroxisomal matrix protein import system shares mechanistic similarities with the endoplasmic reticulum/proteasome degradation process, indicating a common evolutionary history.