Detoxification Of Reactive Carbonyl Species Research Articles

Glyoxal oxidase-mediated detoxification of reactive carbonyl species contributes to virulence, stress tolerance, and development in a pathogenic fungus.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance.

Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of β-carotene and, consequently, β-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing β-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from β-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and β-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from β-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Protection of Polyphenols against Glyco-Oxidative Stress: Involvement of Glyoxalase Pathway.

Chronic high glucose (HG) exposure increases methylglyoxal (MGO)-derived advanced glycation end-products (AGEs) and is involved in the onset of pathological conditions, such as diabetes, atherosclerosis and chronic-degenerative diseases. Under physiologic conditions the harmful effects of MGO are contrasted by glyoxalase system that is implicated in the detoxification of Reactive Carbonyl Species (RCS) and maintain the homeostasis of the redox environment of the cell. Polyphenols are the most abundant antioxidants in the diet and present various health benefits. Aims of the study were to investigate the effects of HG-chronic exposure on glyco-oxidation and glyoxalase system in intestinal cells, using CaCo-2 cells. Moreover, we studied the effect of apple polyphenols on glyco-oxidative stress. Our data demonstrated that HG-treatment triggers glyco-oxidation stress with a significant increase in intracellular Reactive Oxygen Species (ROS), lipid peroxidation, AGEs, and increase of Glyoxalase I (GlxI) activity. On the contrary, Glyoxalase II (GlxII) activity was lower in HG-treated cells. We demonstrate that apple polyphenols exert a protective effect against oxidative stress and dicarbonyl stress. The increase of total antioxidant capacity and glutathione (GSH) levels in HG-treated cells in the presence of apple polyphenols was associated with a decrease of GlxI activity.

PpAKR1A, a Novel Aldo-Keto Reductase from Physcomitrella Patens, Plays a Positive Role in Salt Stress.

The moss Physcomitrella patens is tolerant of highly saline environments. In plants, salinity stress may induce the production of toxic reactive carbonyl species (RCS) and oxidative damage. Aldo-keto reductases (AKRs) are a large group of NADP-dependent oxidoreductases involved in RCS detoxification. However, many members in this superfamily remain uncharacterized. In this study, we cloned and characterised a putative AKR1 from P. patens, named PpAKR1A. Notably, the transcription level of PpAKR1A was induced by salt and methylglyoxal (MG) stress, and the recombinant PpAKR1A protein catalysed the reduction of toxic aldehydes. PpAKR1A knockout mutants of P. patens (ppakr1a) were sensitive to NaCl and MG treatment, as indicated by much lower concentrations of chlorophyll and much higher concentrations of MG and H2O2 than those in WT plants. Meanwhile, ppakr1a plants exhibited decreases in the MG-reducing activity and reactive oxygen species-scavenging ability in response to salt stress, possibly due to decreases in the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Our results indicate that PpAKR1A is an aldo-keto reductase that detoxifies MG and thus plays an important role in salt stress tolerance in P. patens.

Detoxification of Reactive Carbonyl Species by Glutathione Transferase Tau Isozymes.

Oxidative stimuli to living cells results in the formation of lipid peroxides, from which various aldehydes and ketones (oxylipin carbonyls) are inevitably produced. Among the oxylipin carbonyls, those with an α,β-unsaturated bond are designated as reactive carbonyl species (RCS) because they have high electrophilicity and biological activity. Plants have arrays of dehydrogenases and reductases to metabolize a variety of RCS that occur in the cells, but these enzymes are not efficient to scavenge the most toxic RCS (i.e., acrolein) because they have only low affinity. Two glutathione transferase (GST) isozymes belonging to the plant-specific Tau class were recently observed to scavenge acrolein with KM values at a submillimolar level. This suggests that GST could also be involved in the defense system against RCS. We tested the activities of 23 Tau isozymes of Arabidopsis thaliana for five types of RCS, and the results revealed that 11 isozymes recognized either acrolein or 4-hydroxy-(E)-2-nonenal or both as a substrate(s). Such RCS-scavenging activities indicate the potential contribution of GST to RCS scavenging in plants, and they may account for the stress tolerance conferred by several Tau isozymes. RCS are therefore a strong candidate for endogenous substrates of plant GSTs.

Dissecting the Physiological Function of Plant Glyoxalase I and Glyoxalase I-Like Proteins.

The Arabidopsis genome annotation include 11 glyoxalase I (GLXI) genes, all encoding for protein members of the vicinal oxygen chelate (VOC) superfamily. The biochemical properties and physiological importance of three Arabidopsis GLXI proteins in the detoxification of reactive carbonyl species has been recently described. Analyses of phylogenetic relationships and conserved GLXI binding sites indicate that the other eight GLXI genes (GLXI-like) do not encode for proteins with GLXI activity. In this perspective article we analyse the structural features of GLXI and GLXI-like proteins, and explore splice forms and transcript abundance under abiotic stress conditions. Finally, we discuss future directions of research on this topic with respect to the substrate identification of GLXI and GLXI-like proteins and the need of reliable quantitative measurements of reactive carbonyl species in plant tissues.

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5βR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5βR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5βR). The three enzymes show a high degree of sequence and structure conservation in the active site. Amino acids previously considered to allow discrimination between progesterone 5β-reductase and iridoid synthase were interchanged among rAtSt5βR, rCrISY and rDlP5βR applying site-directed mutagenesis. Structural homologous substitutions had different effects, and changes in progesterone 5β-reductase and iridoid synthase activity were not correlated in all cases. Our results help to explain fortuitous emergence of metabolic pathways and product accumulation. The fact that PRISEs are found ubiquitously in spermatophytes insinuates that PRISEs might have a more general function in plant metabolism such as, for example, the detoxification of reactive carbonyl species.

High level of reduced glutathione contributes to detoxification of lipid peroxide-derived reactive carbonyl species in transgenic Arabidopsis overexpressing glutathione reductase under aluminum stress.

Lipid peroxide-derived reactive carbonyl species (RCS), generated downstream of reactive oxygen species (ROS), are critical damage-inducing species in plant aluminum (Al) toxicity. In mammals, RCS are scavenged primarily by glutathione (reduced form of glutathione, GSH), but in plant Al stress, contribution of GSH to RCS detoxification has not been evaluated. In this study, Arabidopsis plants overexpressing the gene AtGR1 (accession code At3g24170), encoding glutathione reductase (GR), were generated, and their performance under Al stress was examined. These transgenic plants (GR-OE plants) showed higher GSH levels and GSH/GSSG (oxidized form of GSH) ratio, and an improved Al tolerance as they suffered less inhibition of root growth than wild-type under Al stress. Exogenous application of 4-hydroxy-2-nonenal, an RCS responsible for Al toxicity in roots, markedly inhibited root growth in wild-type plants. GR-OE plants suffered significantly smaller inhibition, indicating that the enhanced GSH level increased the capacity of RCS detoxification. The generation of H2 O2 due to Al stress in GR-OE plants was lower by 26% than in wild-type. Levels of various RCS, such as malondialdehyde, butyraldehyde, phenylacetaldehyde, (E)-2-heptenal and n-octanal, were suppressed by more than 50%. These results indicate that high levels of GSH and GSH/GSSG ratio by GR overexpression contributed to the suppression of not only ROS, but also RCS. Thus, the maintenance of GSH level by overexpressing GR reinforces dual detoxification functions in plants and is an efficient approach to enhance Al tolerance.

Oxidative and reductive metabolism of lipid-peroxidation derived carbonyls

Extensive research has shown that increased production of reactive oxygen species (ROS) results in tissue injury under a variety of pathological conditions and chronic degenerative diseases. While ROS are highly reactive and can incite significant injury, polyunsaturated lipids in membranes and lipoproteins are their main targets. ROS-triggered lipid-peroxidation reactions generate a range of reactive carbonyl species (RCS), and these RCS spread and amplify ROS-related injury. Several RCS generated in oxidizing lipids, such as 4-hydroxy trans-2-nonenal (HNE), 4-oxo-2-(E)-nonenal (ONE), acrolein, malondialdehyde (MDA) and phospholipid aldehydes have been shown to be produced under conditions of oxidative stress and contribute to tissue injury and dysfunction by depleting glutathione and other reductants leading to the modification of proteins, lipids, and DNA. To prevent tissue injury, these RCS are metabolized by several oxidoreductases, including members of the aldo–keto reductase (AKR) superfamily, aldehyde dehydrogenases (ALDHs), and alcohol dehydrogenases (ADHs). Metabolism via these enzymes results in RCS inactivation and detoxification, although under some conditions, it can also lead to the generation of signaling molecules that trigger adaptive responses. Metabolic transformation and detoxification of RCS by oxidoreductases prevent indiscriminate ROS toxicity, while at the same time, preserving ROS signaling. A better understanding of RCS metabolism by oxidoreductases could lead to the development of novel therapeutic interventions to decrease oxidative injury in several disease states and to enhance resistance to ROS-induced toxicity.

Identification of glutathione (GSH)-independent glyoxalase III from Schizosaccharomyces pombe.

BackgroundReactive carbonyl species (RCS), such as methylglyoxal (MG) and glyoxal (GO), are synthesized as toxic metabolites in living systems. Mechanisms of RCS detoxification include the glutathione (GSH)-dependent system consisting of glyoxalase I (GLO1) and glyoxalase II (GLO2), and GSH-independent system involving glyoxalase III (GLO3). Hsp31 and DJ-1 proteins are weakly homologous to each other and belong to two different subfamilies of the DJ-1/Hsp31/PfpI superfamily. Recently, the Escherichia coli Hsp31 protein and the DJ-1 proteins from Arabidopsis thaliana and metazoans have been demonstrated to have GLO3 activity.ResultsWe performed a systematic survey of homologs of DJ-1 and Hsp31 in fungi. We found that DJ-1 proteins have a very limited distribution in fungi, whereas Hsp31 proteins are widely distributed among different fungal groups. Phylogenetic analysis revealed that fungal and metazoan DJ-1 proteins and bacterial YajL proteins are most closely related and together form a sister clade to bacterial and fungal Hsp31 proteins. We showed that two Schizosaccharomyces pombe Hsp31 proteins (Hsp3101 and Hsp3102) and one Saccharomyces cerevisiae Hsp31 protein (ScHsp31) displayed significantly higher in vitro GLO3 activity than S. pombe DJ-1 (SpDJ-1). Overexpression of hsp3101, hsp3102 and ScHSP31 could confer MG and GO resistance on either wild-type S. pombe cells or GLO1 deletion of S. pombe. S. pombe DJ-1 and Hsp31 proteins exhibit different patterns of subcellular localization.ConclusionsOur results suggest that fungal Hsp31 proteins are the major GLO3 that may have some role in protecting cells from RCS toxicity in fungi. Our results also support the view that the GLO3 activity of Hsp31 proteins may have evolved independently from that of DJ-1 proteins.

Role of oxidative stress in diabetic complications: a new perspective on an old paradigm.

Oxidative stress and oxidative damage to tissues are common end points of chronic diseases, such as atherosclerosis, diabetes, and rheumatoid arthritis. The question addressed in this review is whether increased oxidative stress has a primary role in the pathogenesis of diabetic complications or whether it is a secondary indicator of end-stage tissue damage in diabetes. The increase in glycoxidation and lipoxidation products in plasma and tissue proteins suggests that oxidative stress is increased in diabetes. However, some of these products, such as 3-deoxyglucosone adducts to lysine and arginine residues, are formed independent of oxidation chemistry. Elevated levels of oxidizable substrates may also explain the increase in glycoxidation and lipoxidation products in tissue proteins, without the necessity of invoking an increase in oxidative stress. Further, age-adjusted levels of oxidized amino acids, a more direct indicator of oxidative stress, are not increased in skin collagen in diabetes. We propose that the increased chemical modification of proteins by carbohydrates and lipids in diabetes is the result of overload on metabolic pathways involved in detoxification of reactive carbonyl species, leading to a general increase in steady-state levels of reactive carbonyl compounds formed by both oxidative and nonoxidative reactions. The increase in glycoxidation and lipoxidation of tissue proteins in diabetes may therefore be viewed as the result of increased carbonyl stress. The distinction between oxidative and carbonyl stress is discussed along with the therapeutic implications of this difference.