The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD⁺ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.     

Biogenesis of peroxisomes and mitochondria: linked by division

Peroxisomes and mitochondria are metabolically linked organelles, which are crucial to human health and development. The search for components involved in their dynamics and maintenance led to the interesting finding that mitochondria and peroxisomes share components of their division machinery. Recently, it became clear that this is a common strategy used by mammals, fungi and plants. Furthermore, a closer interrelationship between peroxisomes and mitochondria has been proposed, which might have an impact on functionality and disease conditions. Here, we briefly highlight the major findings, views and open questions concerning peroxisomal formation, division, and interrelationship with mitochondria. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.     

Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.     

Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism

Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult mouse (C57BL/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic as well as biochemical techniques and by the use of various peroxisomal marker proteins (Pex13p, Pex14p, ABCD3, beta-oxidation enzymes and catalase). In contrast to previous reports, we have found that peroxisomes are present in all cell types in human and mouse lungs. However, they differ significantly and in a cell-type-specific manner in their structure, numerical abundance and enzyme composition. Whereas catalase showed significant differences between distinct cell types, Pex14p proved to be the marker of choice for labeling all lung peroxisomes. In alveolar type II cells and alveolar macrophages peroxisomes contained significant amounts of the lipid transporter ABCD3 and beta-oxidation enzymes, suggesting their involvement in the modification and recycling of surfactant lipids and in the control of lipid mediators and ligands for nuclear receptors of the PPAR family. Possible connections between ROS and lipid metabolism of lung peroxisomes are discussed.     

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and β-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Peroxisome through cell differentiation and neoplasia

Summary— Peroxisomes are essential in cellular metabolism as their dysgenesis or defects in single enzymes or impairment of multiple peroxisomal enzymatic functions have been found in several inherited metabolic diseases with serious clinical sequelae. The assembly and formation of these cytoplasmic organelles constitute a major and intringuing research topic. In the present study the biogenesis of peroxisomes and the developmental patterns of their enzymes have been reviewed during embryonic and/or post-embryonic ontogenesis of lower (amphibians) and higher (avians, mammals) vertebrates. In developing vertebrates, epithelial cell differentiation is accompanied by increases in frequency and size of peroxisomes. The tissue-specific expression of peroxisomal enzymes contributes substantially to the biochemical maturation of epithelial cells. The relationship between biogenesis of peroxisomes, expression of peroxisomal enzymes and structural and functional cellular phenotype has also been investigated in differentiating epithelial cells along the crypt-villus axis of the adult rat intestine. Cytochemical studies at the ultrastructural level have provided evidence that peroxisomes are already present in proliferating cells of the intestinal crypt region before they begin to differentiate. Migration and differentiation of intestinal epithelial cells from crypt to villus compartments are marked by significant increases in number and size of catalase-positive structures. Increasing activity gradients from crypt to surface areas are found for the peroxisomal oxidases examined (enzymes of the peroxisomal β-oxidation system, d -amino acid oxidase and polyamine oxidase). Thus, peroxisomes are more and more involved in oxidative metabolic pathways as intestinal epithelial cells differentiate. Finally, we have analyzed the peroxisomal behaviour in human neoplastic epithelial cells. The presence of peroxisomes has been cytochemically revealed in human breast and colon carcinomas. Peroxisomal enzyme specific activities are significantly lower in human breast and colon carcinomas than in the adjacent healthy mucosa. Furthermore, a relationship is found between the specific activities of some peroxisomal enzymes and the histological tumour grades.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Hypocatalasemic fibroblasts accumulate hydrogen peroxide and display age-associated pathologies

Human epidemiological studies point to an association of hypocatalasemia and an increased risk of age-related disease. Unfortunately, the cellular and molecular manifestations of hypocatalasemia are only poorly understood. In this analysis, we have extensively characterized hypocatalasemic human fibroblasts and report that they amass hydrogen peroxide and are oxidatively damaged. Protein and DNA alike are affected, as are functioning and biogenesis of peroxisomes - the subcellular organelles which normally house catalase. Despite these pathologies and their relative inability to grow, the cells do not appear to be intrinsically senescent. With the goal of restoring oxidative balance and perhaps reversing some of the accumulated damage to critical cellular components, we transduced hypocatalasemic fibroblasts with a form of catalase specifically designed to efficiently traffic to peroxisomes. We show the strategy is extremely effective, with dramatic reductions seen in cellular hydrogen peroxide levels. Future longitudinal studies aimed at examining the effects of a more continuous and long-term protein therapy may now commence.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Dynamics of peroxisome assembly and function

Recent studies in human cells and in the yeast Yarrowia lipolytica have shown that peroxisomes consist of numerous structurally distinct subcompartments that differ in their import competency for various proteins and are related through a time-ordered conversion of one subcompartment to another. Our studies have implicated the fusion of small peroxisomal precursors as an early event in the multistep assembly of peroxisomes operating in Y. lipolytica. Newly discovered unexpected roles for peroxisomes in specific developmental programs have expanded the remarkable plasticity of peroxisomal functions. Here, we highlight recent discoveries on the highly dynamic nature of peroxisome assembly and function and suggest questions for future research in these areas.     

The peroxisome: an update on mysteries

Peroxisomes contribute to several crucial metabolic processes such as β-oxidation of fatty acids, biosynthesis of ether phospholipids and metabolism of reactive oxygen species, which render them indispensable to human health and development. Peroxisomes are highly dynamic organelles that rapidly assemble, multiply and degrade in response to metabolic needs. In recent years, the interest in peroxisomes and their physiological functions has significantly increased. This review intends to highlight recent discoveries and trends in peroxisome research, and represents an update as well as a continuation of a former review article. Novel exciting findings on the biological functions, 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 are addressed. Furthermore, recent findings on the role of peroxisomes in the brain are discussed.     

Three-dimensional ultrastructural analysis of peroxisomes in HepG2 cells

Peroxisomes in the human hepatoblastoma cell line, HepG2, exhibit distinct alterations of shape, size, and distribution, dependent on culture conditions (cell density, duration in culture, and presence of specific growth factors). Although many cells with elongated tubular peroxisomes are present in thinly seeded cultures, spherical particles forming large focal clusters are found in confluent cultures. The authors have analyzed the ultrastructure and the spatial relationship of peroxisomes of HepG2 cells at different stages of differentiation, using three-dimensional (3D)-reconstruction of ultrathin serial sections, and electronic image processing. Cells were prepared for immunofluorescence using different antibodies against peroxisomal matrix and membrane proteins, as well as for electron microscopy after the alkaline 3,3′-diaminobenzidine staining for catalase. The results indicate that the tubular peroxisomes, which can reach a length of several microns, are consistently isolated, and never form an interconnected peroxisomal reticulum. At the time of disappearance of tubular peroxisomes, rows of spherical peroxisomes, arranged like beads on a string, are observed, suggesting fission of tubular ones. In differentiated confluent cultures, clusters of several peroxisomes are seen, which, by immunofluorescence, appear as large aggregates, but after 3D reconstruction consist of single spherical and angular peroxisomes without interconnections. The majority of such mature spherical peroxisomes (but not the tubular ones) exhibit tail-like, small tubular and vesicular attachments to their surface, suggesting a close functional interaction with neighboring organelles, particularly the endoplasmic reticulum, which is often observed in close vicinity of such peroxisomes.     

Catalase-less peroxisomes. Implication in the milder forms of peroxisome biogenesis disorder

We established a Chinese hamster ovary cell line having a temperature-sensitive phenotype in peroxisome biogenesis. This mutant (65TS) was produced by transforming a PEX2-defective mutant, Z65, with a mutant PEX2 gene, PEX2(E55K), derived from a patient with infantile Refsum disease, a milder form of peroxisome biogenesis disorder. In 65TS, catalase was found in the cytosol at a nonpermissive temperature (39 degrees C), but upon the shift to a permissive temperature (33 degrees C), catalase gradually localized to the structures containing a 70-kDa peroxisomal membrane protein, PMP70. In contrast to catalase, other matrix proteins containing typical peroxisome targeting signals, acyl-CoA oxidase and peroxisomal 3-ketoacyl-CoA thiolase, were co-localized with PMP70 in most cells, even at 39 degrees C. We found that these structures are partially functional peroxisomes and named them "catalase-less peroxisomes." Catalase-less peroxisomes were also observed in human fibroblasts from patients with milder forms of peroxisome biogenesis disorder, including the one from which the mutant PEX2 gene was derived. We suggest that these structures are the causes of the milder phenotypes of the patients. Temperature-dependent restoration of the peroxisomes in 65TS occurred even in the presence of cycloheximide, a protein synthesis inhibitor. Thus, we conclude that in 65TS, catalase-less peroxisomes are the direct precursors of peroxisomes.     

Versatility of peroxisomes: An evolving concept

Research spanning almost 50 years has highlighted unique characteristics and irreplaceable list of diverse functions performed by peroxisomes in various model systems. Peroxisomes are single membrane bound highly dynamic organelles ubiquitous to most eukaryotic cells. Proliferation by division of pre-existing organelles and the role of endoplasmic reticulum in the biogenesis of these organelles is now well established. The earliest identified conserved functions of peroxisomes are β-oxidation of fatty acids and reactive oxygen species metabolism. Several studies over the last few decades have reported the importance of this organelle and its numerous cell type, tissue and environment-dependent functions. Their role in several aspects of human health and disease is now under investigation. Studies related to peroxisome biology and functions are now also extended to diverse model systems like Drosophila melanogaster, trypanosomatids, etc. Peroxisomes also intricately collaborate and carry out these functions together with several other organelles in a cell. In this review, we aim to present an overview of our current knowledge of the repertoire of functions of peroxisomes in various model systems.     

Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the number of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling.     

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.     

Current cytochemical techniques for the investigation of peroxisomes. A review.

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3'-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of "preperoxisomes" is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins. (J Histochem Cytochem 47:1219-1232, 1999)     

Peroxisomal enzyme activities in the human hepatoblastoma cell line HepG2 as compared to human liver.

In order to develop an in vitro model allowing investigation of the long-term effects of hormones and other agents on peroxisomes in liver cells, we measured the activity of a series of peroxisomal enzyme activities in HepG2 cells, a proliferating cell line derived from a human hepatoblastoma. The results obtained show that although in absolute terms peroxisomal enzyme activities are lower in HepG2 cells as compared to human liver, relative activities were comparable in HepG2 and human liver, respectively. Furthermore, it is shown that peroxisomes can easily be isolated from HepG2 cells using density gradient centrifugation. It is concluded that HepG2 cells represent a good model system to study the characteristic (long-term) regulation and control of metabolism of human liver peroxisomes.