A specific fluorescence probe for hydrogen peroxide detection in peroxisomes

Hydrogen peroxide is an important mediator in cell signalling and cell death. Apart from the mitochondrion the peroxisome is the most important cellular site for the generation and scavenging of hydrogen peroxide. Peroxisomes contain various oxidases, e.g. for the metabolism of long-chain fatty acids, polyamines, and for the oxidation of urate, which form hydrogen peroxide. Widely-used chemical probes for the detection of hydrogen peroxide like dichlorofluorescein diacetate (DCFDA) often lack in specificity and the possibility of compartment-specific measurement. To overcome these disadvantages, Belousov et al. developed the novel hydrogen peroxide sensitive fluorescent protein HyPer. In the present study the HyPer protein was fused with the PTS1 tag for a specific hydrogen peroxide detection in peroxisomes. The localization of the HyPer protein in the peroxisomes was confirmed by immunofluorescence and the functionality by fluorescence microscopy and flow cytometry analyses. The presented HyPer-Peroxi fluorescent protein is a valuable tool for studying hydrogen peroxide generation within the peroxisomes.     

Plant peroxisomes at the crossroad of NO and H_2O_2 metabolism

Plant peroxisomes are subcellular compartments involved in many biochemical pathways during the life cycle of a plant but also in the mechanism of response against adverse environmental conditions. These organelles have an active nitro-oxidative metabolism under physiological conditions but this could be exacerbated under stress situations. Furthermore, peroxisomes have the capacity to proliferateand also undergo biochemical adaptations depending on the surrounding cellular status. An important characteristic of peroxisomes is that they have a dynamic metabolism of reactive nitrogen and oxygen species(RNS and ROS) which generates two key molecules, nitric oxide(NO) and hydrogen peroxide(H_2O_2). These molecules can exert signaling functions by means of post-translational modifications that affect the functionality of target molecules like proteins, peptides or fatty acids. This review provides an overview of the endogenous metabolism of ROS and RNS in peroxisomes with special emphasis on polyamine and uric acid metabolism as well as the possibility that these organelles could be a source of signal molecules involved in the functional interconnection with other subcellular compartments.     

Mammalian peroxisomes and reactive oxygen species

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor- on peroxisome function and peroxisome proliferator activated receptor-. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5^–/– knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.     

Reactive oxygen species, antioxidant systems and nitric oxide in peroxisomes

Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. Like chloroplasts and mitochondria, plant peroxisomes also produce superoxide radicals (O2*(-)) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29 and 32 kDa have been shown to generate radicals O2*(-). Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the ascorbate-glutathione cycle, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of peroxisomes. The four enzymes of the ascorbate-glutathione cycle (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) as well as the antioxidants glutathione and ascorbate have been found in plant peroxisomes. The recycling of NADPH from NADP(+) can be carried out in peroxisomes by three dehydrogenases: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase. In the last decade, different experimental evidence has suggested the existence of cellular functions for peroxisomes related to reactive oxygen species (ROS), but the recent demonstration of the presence of nitric oxide synthase (NOS) in plant peroxisomes implies that these organelles could also have a function in plant cells as a source of signal molecules like nitric oxide (NO*), superoxide radicals, hydrogen peroxide, and possibly S-nitrosoglutathione (GSNO).     

The peroxisome: orchestrating important developmental decisions from inside the cell

The peroxisome has long been known for its role in lipid metabolism and hydrogen peroxide detoxification. However, growing evidence supports the view that this organelle can also function both as an intracellular signaling compartment and as an organizing platform that orchestrates certain developmental decisions from inside the cell. This review highlights various strategies that peroxisomes employ to regulate the processes of development, differentiation, and morphogenesis and critically evaluates several molecular mechanisms by which peroxisomes promote these processes.     

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.     

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.     

Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality

In photosynthetic cells, a large amount of hydrogen peroxide is produced in peroxisomes through photorespiration, which is a metabolic pathway related to photosynthesis. Hydrogen peroxide, a reactive oxygen species, oxidizes peroxisomal proteins and membrane lipids, resulting in a decrease in peroxisomal quality. We demonstrate that the autophagic system is responsible for the elimination of oxidized peroxisomes in plant. We isolated Arabidopsis mutants that accumulated oxidized peroxisomes, which formed large aggregates. We revealed that these mutants were defective in autophagy-related (ATG) genes and that the aggregated peroxisomes were selectively targeted by the autophagic machinery. These findings suggest that autophagy plays an important role in the quality control of peroxisomes by the selective degradation of oxidized peroxisomes. In addition, the results suggest that autophagy is also responsible for the functional transition of glyoxysomes to leaf peroxisomes.     

Lipid metabolism in peroxisomes in relation to human disease

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1, fatty acid β-oxidation 2, fatty acid α-oxidation 3, synthesis of cholesterol and other isoprenoids 4, ether-phospholipid synthesis and 5, biosynthesis of polyunsaturated fatty acids. In Chapter 2–6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attentin is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for trransport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.     

L-Carnitine alters nitric oxide synthase activity in fibroblasts depending on the peroxisomal status

Fibroblast cellular models are widely used for research on fatty acid metabolism. Due to the importance of L-carnitine in intermediary metabolism we studied the effects of L-carnitine on healthy human skin fibroblasts and fibroblasts without functional peroxisomes (Zellweger Syndrome) cultivated under carnitine deficiency, which is caused by standard media compositions. The application of physiological (0.1mM) or super-physiological (1mM) doses of L-carnitine causes a significant decrease of the specific activity of nitric oxide synthase (NOS, 2.25+/-0.10 to 1.36 pmol/(minmg)+/-0.09 pmol/(minmg) at 0.1mM), proliferation and a tendentious decrease of the antioxidant defence potential against hydrogen peroxide only in control cells. Simultaneous application of L-carnitine and 100 micro M N-acetylcysteine (NAC) prevents the alterations in control cells. Thus, L-carnitine alters the cellular regulation of the NOS probably by reactive oxygen species (ROS), which suggests that carnitine deficient media neither reflect physiological conditions for cellular models for fatty acid metabolism nor for the regulation of NOS.     

Hydrogen peroxide probes directed to different cellular compartments

Background

Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings

Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events.

Conclusions

We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells.     

Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.     

Metabolism of oxygen radicals in peroxisomes and cellular implications.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.     

Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation

Seaweeds grow in distinct vertical bands on the seashore and it is well known that their ability to recover physiological processes following desiccation is correlated to their shore position. Despite this, little is known of the cellular mechanisms by which intertidal seaweeds limit membrane damage during desiccation and subsequent rehydration. In this study, specimens of the intertidal red seaweed Stictosiphonia arbuscula were placed in sealed tanks and maintained at different relative humidities (control, RH 90-100%; moderate desiccation, RH 70-80% and severe desiccation, RH 40-50%) for 12, 24 or 48 h. Membrane damage and antioxidant metabolism was examined immediately following specimen rehydration. Amino acid leakage, through the plasmalemma, was greater for desiccated low-band specimens than high-band specimens, indicating greater membrane damage. In addition, low-band specimens produced more hydrogen peroxide and lipid hydroperoxides than high-band specimens. This indicates that, upon rehydration, high-band populations have a greater ability to reduce the build-up of hydrogen peroxide, limit lipid peroxidation and hence membrane and protein damage, than low-band populations. The greater ability to prevent or reduce the production of reactive oxygen species was not due to a larger antioxidant pool, but rather increased activity of the enzymes required to regenerate ascorbate and glutathione. These findings suggest that antioxidant metabolism is one of the defence mechanisms that protect S. arbuscula from cellular damage due to desiccation.     

Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves

Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide,metabolism of fatty acids, photorespiration, and the biosynthesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry(MS)-based proteomics has been performed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of peroxisomes from Arabidopsis leaves given a 48-h dark treatment.Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identifieda total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the ful function of plant peroxisomes.     

The role of peroxisomes in the integration of metabolism and evolutionary diversity of photosynthetic organisms

The peroxisome is a metabolic compartment serving for the rapid oxidation of substrates, a process that is not coupled to energy conservation. In plants and algae, peroxisomes connect biosynthetic and oxidative metabolic routes and compartmentalize potentially lethal steps of metabolism such as the formation of reactive oxygen species and glyoxylate, thus preventing poisoning of the cell and futile recycling. Peroxisomes exhibit properties resembling inside-out vesicles and possess special systems for the import of specific proteins, which form multi-enzyme complexes (metabolons) linking numerous reactions to flavin-dependent oxidation, coupled to the decomposition of hydrogen peroxide by catalase. Hydrogen peroxide and superoxide originating in peroxisomes are important mediators in signal transduction pathways, particularly those involving salicylic acid. By contributing to the synthesis of oxalate, formate and other organic acids, peroxisomes regulate major fluxes of primary and secondary metabolism. The evolutionary diversity of algae has led to the presence of a wide range of enzymes in the peroxisomes that are only similar to higher plants in their direct predecessors, the Charophyceae. The appearance of seed plants was connected to the acquirement by storage tissues, of a peroxisomal fatty acid oxidation function linked to the glyoxylate cycle, which is induced during seed germination and maturation. Rearrangement of the peroxisomal photorespiratory function between different tissues of higher plants led to the appearance of different types of photosynthetic metabolism. The peroxisome may therefore have played a key role in the evolutionary formation of metabolic networks, via establishing interconnections between different metabolic compartments.     

Significance of plant sulfite oxidase

Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants.     

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.     

Identification of human PMP34 as a peroxisomal ATP transporter

In recent years much has been learned about the essential role of peroxisomes in cellular metabolism. Much less, however, is known about the permeability properties of peroxisomes although it is well established now that peroxisomes are impermeable to small molecules which implies the existence of transporters in the peroxisomal membrane. In this paper we report the identification of PMP34, a peroxisomal membrane protein belonging to the mitochondrial solute carrier family, as an adenine nucleotide transporter. This is concluded from different experimental findings including rescue of the defect in medium-chain fatty acid oxidation in Saccharomyces cerevisiae cells in which the ANT1 gene coding for Ant1p, the peroxisomal adenine nucleotide carrier, was disrupted. Furthermore, we have purified PMP34, reconstituted the protein in proteoliposomes, and provide direct proof that PMP34 is an adenine nucleotide transporter.