Peroxisomes are cellular organelles present in the eukaryotic cells that are responsible for detoxification processes such as oxidation of fatty acids, biosynthesis of glycerolipids and isoprenoids, among others. Due to the number of functions which they carry out, they are conditioned to an elevated production of free radicals (FR), for which they have a sophisticated antioxidant system consisting of enzymes that can neutralize free radical molecules. The importance of peroxisomes in the development of age-related diseases such as diabetes, cancer and cardiovascular disease has been reported recently. In this paper, we review briefly these organelles, treating aspects such as their evolution, functions and the role they play in oxidative stress processes associated with aging.
Keywords: ROS, Aging, peroxisomes.
Dr. Rodhan first described peroxisomes in 1951 from mice kidney cells, but they were named microbodies. Later, in 1966, Dr. De Duve1 and his research team isolated the peroxisomes of rat liver cells to study their biochemical properties. They identified a high enzymatic activity involved in the degradation of hydrogen peroxide (H2O2), for which they named them 'peroxisomes'. Since then, researchers started to isolate these organelles from different cell types; it was found that they had a high functional variability, which depends on the specific tissue, the environmental conditions or the organisms from which they were obtained. One example is the glycosomes found in trypanosomatid species, so called because they are peroxisomes that carry out glycolytic activities.2
Peroxisomes are organelles found in a large variety of organisms including eukaryotic cells, plants, yeasts, etc.; in general, a single delimiting membrane, with fairly well organized systems for biogenesis and maintenance, forms most of them. Processes such as fatty acid oxidation and free radical production (FR) are carried out inside these organelles due to the presence of enzymes that catalyze such reactions. Unlike the mitochondria and the cell nucleus, peroxisomes do not have their own genetic material; peroxisomal proteins are encoded in the nucleus and are imported by peroxisomes via specific routes dependent on targeting signals. Most peroxisomal matrix proteins use small peroxisomal targeting signals (PTS) on their C-terminal side which basically consist of three amino acids: serine-lysine-leucine (SKL), or of amino acid residues involved with transport.3 Other peroxisomal proteins are not able to recognize the PTS and instead are transported to the peroxisomes on the Pex5 domains or on protein carriers of PTS. The targeting signals are recognized by the molecular systems, allowing the mobilization of proteins within the peroxisomal matrix. This complex or importomer4 consists of two basic parts: a membrane complex which includes the protein receptor PEX13 and Pex14, and a receptor export module in the cytoplasm containing different proteins with ring domains, ubiquitination enzymes and the AAA-ATPases Pex1 and Pex6.5 This system is used by protein receptors such as Pex5 and PEX7, which transport in and out of the organelle, importing loads to the peroxisomal matrix. The import machinery of peroxisomes is completely different to that of other organelles such as mitochondria or chloroplasts, but is capable of transporting folded proteins and even some oligomers. A characteristic which the peroxisomes of different species appear to share is the division of the organelle. The machinery responsible for the division consists of at least one dynein protein and a TPR protein (Tetratrico Peptide Repeat) with motifs that bind to the membrane. These proteins are also involved in mitochondrial fission, establishing an interconnection between these organelles.6
Recently, it was documented that deficiency of peroxisomes in neuronal cells (Nestin-Pex5/) of mice is associated with the development of neurodegenerative phenotypes that lead to cognitive and motor disorders and that in advanced stages progress to early death. Among the pathological phenomena observed are demyelination, axonal degeneration and neuroinflammation. Additionally, has been studied the role of the deficiency of plasmalogens such as Gnpat/, which is related to demyelination in Purkinje cells and axonal damage in the brain; another plasmalogen is CMV-Tx-Pex5/, associated with demyelination of the cerebellum and corpus callosum, suggesting that peroxisomal inactivity, coupled with the decrease of plasmalogens, triggers a quick inflammatory response, demyelination, axonal damage and loss.7
There are different theories about the origin of peroxisomes as cell organelles. Shortly after their discovery, a close relationship with the Endoplasmic Reticulum (RE) was observed through the micrographs, which suggested that peroxisomes were formed from the endomembrane system. Afterwards, it was found that new peroxisomes are formed from existing ones, and so an endosymbiotic origin was proposed. In 1982, da Duve proposed a model in which he explains a scenario with conditions in which the enzymes participate in the metabolism and detoxification of hydrogen peroxide (H2O2). According to this model, is speculated that atmospheric levels of oxygen proved toxic to most living organisms, so the cells had to develop defense systems to survive under these conditions. This has been one of the most widely accepted theories by biologists. However, the relationship between peroxisome biogenesis and RE has been considered again in recent years. The reason is that it has been observed that some membrane proteins of peroxisomes (PMP) must head to the RE before reaching the peroxisomes.8 Phylogenetic studies provide further evidence of the homologous relationship between the importation of some components of the peroxisome machinery and the components associated to the RE pathways, fueling the doubts about the endosymbiotic origin of the enzyme systems of the matrix.9
The high enzymatic activity of peroxisomes is a product of the inherent evolution of the organisms and is thought to be the result of the selective acquisition of enzymes and full paths; one example is that of alanine-glyoxylate aminotransferase, which is redirected to the peroxisomes in different lineages of mammals due to changes in their eating habits.10 All this evidence supports the idea that the diverse metabolic activity of peroxisomes is due to the incorporation of different proteins. Thus, peroxisomes are considered to possess a highly modifiable proteome the metabolic potential of which has adapted to the conditions and needs of each species.
A free radical (FR) is any atom or group of atoms with one or more unpaired electrons; they travel throughout the body trying to steal an electron from the stable molecules in order to achieve electrochemical stability. Once the FR has managed to steal the electron it needs, the stable molecule that ceded it becomes an FR with an unpaired electron, so that this event becomes a true chain reaction. The biological activity of the FR lasts a very short time; however, they have an impressive ability to react to what is around them, destabilizing molecules and damaging cell membranes.11 The body generates FR in a natural way in response to a large number of reactions and to the presence of bacteria and viruses; the body has its own mechanisms to neutralize naturally produced free radicals; the endogenous enzyme systems catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) take care of them. External environmental conditions, diet, excessive exercise, pollution, consumption of tobacco and alcohol, etc.., expose the body to a large number of FR. Reactive Oxygen Species (ROS) are oxygen compounds that are continuously produced in the organism through redox reactions; they have toxic activity and are believed to be responsible for the damage to macromolecules such as DNA, lipids, carbohydrates and proteins. The best known ROS are the superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-).11, 12
In plants and in most eukaryotic cells, peroxisomes are organelles in which there is an increased production of H2O2. In different types of peroxisomes, the main metabolic process responsible for the production of H2O2 is the β-oxidation of fatty acids, the photorespiratory reaction glycolate oxidase, the enzymatic reaction of flavin oxidase and the disproportionation of O2- radicals. Peroxisomes, like mitochondria and chloroplasts, produce O2- radicals as a result of their normal metabolism. In peroxisomes, the membrane is where the radicals O2- are produced; a small flow of electrons seems to be involved, as well as a flavoprotein NADH, ferricyanide reductase, and a cytochrome b.13 A series of membrane proteins of 18, 29 and 32 kD have been identified in the peroxisomes of pea leaves; these proteins are responsible for the production of radicals O2-.Of them, the 18 kD protein is the one that produces the highest levels of O2- and has been identified as a cytochrome b.
As mentioned above, the mitochondria is the main responsible for the formation of ROS; however, in peroxisomes, the amino acid oxidase, the acyl-CoA oxidase and the urate oxidase generate H2O2; this molecule, like the radical O2-, does not show a high reactivity to macromolecules, but in the presence of certain transition metal complexes such as iron or copper it can react with other H2O2 molecules through the Fenton or Haber-Weiss reaction and produce the OH- radical, which is highly reactive. We have natural cellular antioxidant defense mechanisms that regulate the generation of these substances, either by reducing them or by sequestering them. In the peroxisomes, the main defense system are catalase, SOD, Cu, Zn and peroxiredoxin 5 (PRDX5).14 In the case of certain pathologies or the aging process, these defense systems become insufficient to counteract the excessive production of ROS, which generates an oxidative stress that induces damage to DNA, cellular membrane lipids and proteins.15 It is also known that during the cellular aging process, structural changes occur in the enzymes due to translation errors that affect the conformation and sequencing of aminoacids, glycosylation and oxidation.
It is known that the production of H2O2 causes molecular damage that can induce the reduction of the rates of division through the pathway of the extracellular mitogenic factor regulated by protein kinase ½ ("Erk ½"), which is known to be sensitive to the presence of H2O2 (Kim, et al., 2003)16. In contrast, it is known that normal levels of ROS production serve as intracellular signaling for cell proliferation and differentiation reactions, and also of inflammatory reactions and immune responses.17
Moreover, it has been reported that the production of ROS in peroxisomes affects the redox balance in the mitochondria, which can lead to excessive fragmentation of the mitochondria; this suggests that peroxisomes could act as mediators in mitochondrial signaling for the production of ROS; this finding reinforces the view that peroxisomes are not just metabolic organelles, but that they participate as signaling intracellular compartments.18, 19 The mitochondria of cells without catalase experience a loss of the capacity to maintain the redox balance; however, an efficient system for metabolizing ROS or for decreasing its production can be observed in the peroxisomes of these same cells.18 The mechanisms by which such system works are not yet fully elucidated but it is believed that mammalian catalases lack mitochondrial recognition and orientation sequences. Furthermore, H2O2 diffuses freely through the cell membrane; it is speculated that the H2O2 released by the mitochondria is directly degraded by peroxisomes20, although it is well known that the intramitochondrial production of H2O2 is mediated by SODCuZn and PRDX5. The development of innovative biomarkers such as roGFP18 and HyPeR21 has allowed knowing more precisely the role of H2O2 in signaling processes, in the maintaining of homeostasis and in metabolic processes. One example of this is the determination of the toxicity induced by non-esterified free fatty acids in rat pancreatic beta cells, on which HyPer was used as fluorescent marker to detect the production of H2O2; a significant increase in the levels of this radical, due to the processes of peroxisomal β-oxidation, was observed in this study.22
Studies on aging and peroxisomes have been conducted in rat liver cells, in which a decrease in enzyme activity and in the peroxisomal function was associated in general with aging. Some reports confirm the decrease in the activity of the enzymes involved in the β-oxidation of fatty acids and of catalase in old liver cells. This relationship was suggested by the decrease of the PPAR-α (peroxisome proliferator-activated receptor alpha) of RXR-α (Retinoid X receptor alpha), which are thought to be responsible for regulating these systems.23
As mentioned above, most peroxisomal enzymes function with the help of PTS based on the amino acid sequence SLK that determines the capacity of these enzymes to be recognized by the receptor Pex5.The decreased effectiveness of the import of proteins linked to PTS-SKL in senescent cells was found in in vitro tests. Surprisingly, this deficiency was first observed in middle-aged cells and was exacerbated in mature and senescent cells.24
With respect to catalase, the most abundant antioxidant enzyme in peroxisomes, and its decrease in activity due to aging, it has been proposed that subjects with an innate deficiency in the production of this enzyme have an increased risk of suffering diseases related with advanced age such as diabetes, atherosclerosis and cancer.25 It has also been found that, with increasing age, this enzyme becomes a poor substrate for the import system of peroxisomes.15 Catalase is an enzyme containing PTS, but its amino acid sequence consists of a tetrapeptide: lysine-alanine-asparagine-leucine (KANL); this sequence is a weak PTS, even in young cells, which severely compromises the import of catalase into old cells.
An important aspect for the efficient import of proteins is the activity of the Pex5p receptor. It has been reported that the Pex5p receptor has a greater affinity for the SKL sequence found in some proteins than for the KANL sequence present in catalase. Even the change of a KANL sequence for a SKL sequence in catalase promotes the recognition by Pex5p and improves its import to the peroxisome.15 Thereby, it is suggested that during aging there is a decrease in the affinity of Pex5p for its ligand SKL. One of the theories that attempts to explain this says that the incapacity of old cells to remove excess H2O2 and other FR causes oxidative damage to Pex5p in this medium.26, 27
There are chaperone molecules involved in binding Pex5p with SKL such as the Hsp70 protein (stress protein) which also supports the import of PTS; the decrease of the expression, or the absence of Hsp70, has been associated with the aging processes of the peroxisome.28
Some studies have shown that there is stagnation of SKL-containing proteins and of catalase in the cytosol of senescent cells. With aging, the regulatory mechanisms of peroxisome biogenesis become altered, including the formation of the membrane, the import of proteins, growth and division.15 It has been shown in fibroblasts of human skin that aging changes the peroxisomal, mitochondrial and cytosolic redox levels to a more oxidized state, which is consistent with the theory about the increase of oxidative stress with age and the decrease of the antioxidant defense systems.18
Peroxisomes are cellular organelles that have an important role in the production of H2O2, a molecule that can be a precursor of the highly reactive OH radical- and that can cause cell damage at the level of DNA, lipids and proteins. In the young cells, the mechanisms that regulate the production and elimination of ROS are equilibrated by a part of the peroxisomes; however, during the aging process these systems deteriorate. Several studies clearly show the decrease of the enzymes involved in fatty acid oxidation, the reduced efficiency of catalase to eliminate H2O2, the decreased effectiveness of receptors, particularly Pex5p, to bind to their ligand, the low activity of the chaperone molecules Hsp70 that favor the binding of Pex5p to PTS, and the alteration of some other mechanisms. It is important to encourage the scientific study of peroxisomes, of the pathways involved in the production of free radicals and of the development of new biomarkers that allow to determine these pathways; in this way we could consider alternatives that help mediate or reduce excess oxidative stress and therefore the damage done by it to biological systems; and we could also help in delaying aging, an issue that has been much addressed in recent years.
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1Academic Area of Nutrition, Institute of Health Sciences, UAEH, Pachuca, Hgo
2Academic Area of Medicine, Institute of Health Sciences, UAEH, Pachuca, Hgo.
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