Mitochondria-derived hydrogen peroxide selectively enhances T cell receptor-initiated signal transduction.
ABSTRACT: T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O2?(-)) and hydrogen peroxide (H2O2), as second messengers. Although H2O2 can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H2O2 as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O2?(-) into H2O2 and thus acts as an intracellular generator of H2O2. As charged O2?(-) is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O2?(-) leading to production of H2O2. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H2O2 but does not alter the levels of intracellular H2O2 scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLC?1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H2O2 specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H2O2 required to selectively modulate downstream signal transduction pathways.
Project description:This study presents an e.s.r. assay for superoxide dismutase (SOD). Enzymic reactions were studied in which Cu,Zn-SOD, Mn-SOD and Fe-SOD each competed with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) for superoxide anion (O2-) at pH 7.8 O2- from dissolved KO2 (potassium superoxide) in dimethyl sulphoxide was added directly to the enzyme solutions containing DMPO. The results show that, in this competition reaction system, the kinetics of the reactions between the enzymes and O2- follow a function y = f[( SOD]0.5). The rate constant, kSOD = 6.4 x 10(9) M-1. S-1, determined for Cu,Zn-SOD is approximately an order of magnitude larger than those for Mn-SOD and Fe-SOD. A comparative study of reported SOD mimics, including Mn2+, MnO2-desferrioxamine mesylate (Desferal) and MnO2-Desferal-ascorbate, was done. The results show that solutions of these complexes are approximately three orders of magnitude less active than Cu,Zn-SOD and approximately two orders of magnitude less active than Mn-SOD or Fe-SOD. The results also suggest that the reactivity toward O2- in solutions of these complexes originates from the Mn2+ present and not from the MnO2-Desferal complexes.
Project description:Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity capable of inactivating the superoxide anion (•O2-) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34-44?kDa and with very different AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50?kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60?kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2- is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress.
Project description:Superoxide dismutase (SOD) is a very important reactive oxygen species (ROS)-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD), from Sedum alfredii, a cadmium (Cd)/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT) plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2) and superoxide radicals (O2•-). The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress.
Project description:Reactive sulfur species (RSS) such as H2S, HS•, H2Sn, (n = 2-7) and HS2•- are chemically similar to H2O and the reactive oxygen species (ROS) HO•, H2O2, O2•- and act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved H2S, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (H2S2-H2S5). H2S was concentration- and oxygen-dependently oxidized by 1?M SOD to polysulfides (mainly H2S2, and to a lesser extent H2S3 and H2S5) with an EC50 of approximately 380?M H2S. H2S concentrations > 750?M inhibited SOD oxidation (IC50 = 1.25mM) with complete inhibition when H2S > 1.75mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved H2S over hydrosulfide anion (HS-), whereas HS- inhibited polysulfide production. In hypoxia, other possible electron donors such as nitrate, nitrite, sulfite, sulfate, thiosulfate and metabisulfite were ineffective. Manganese SOD also catalyzed H2S oxidation to form polysulfides, but did not metabolize polysulfides indicating common attributes of these SODs. These experiments suggest that, unlike the well-known SOD-mediated dismutation of two O2•- to form H2O2 and O2, SOD catalyzes a reaction using H2S and O2 to form persulfide. These can then combine in various ways to form polysulfides and sulfur oxides. It is also possible that H2S (or polysulfides) interact/react with SOD cysteines to affect catalytic activity or to directly contribute to sulfide metabolism. Our studies suggest that H2S metabolism by SOD may have been an ancient mechanism to detoxify sulfide or to regulate RSS and along with catalase may continue to do so in contemporary organisms.
Project description:Superoxide dismutases play an important role in human health and disease. Three decades of effort have gone into synthesizing SOD mimics for clinical use. The result is the Mn porphyrins which have SOD-like activity. Several clinical trials are underway to test the efficacy of these compounds in patients, particularly as radioprotectors of normal tissue during cancer treatment. However, aqueous chemistry data indicate that the Mn porphyrins react equally well with multiple redox active species in cells including H2O2, O2•-, ONOO-, thiols, and ascorbate among others. The redox potential of the Mn porphyrins is midway between the potentials for the oxidation and reduction of O2•-. This positions them to react equally well as oxidants and reductants in cells. The result of this unique chemistry is that: 1) the species the Mn porphyrins react with in vivo will depend on the relative concentrations of the reactive species and Mn porphyrins in the cell of interest, and 2) the Mn porphyrins will act as catalytic (redox cycling) agents in vivo. The ability of the Mn porphyrins to catalyze protein S-glutathionylation means that Mn porphyrins have the potential to globally modulate cellular redox regulatory signaling networks. The purpose of this review is to summarize the data that indicate the Mn porphyrins have diverse reactions in vivo that are the basis of the observed biological effects. The ability to catalyze multiple reactions in vivo expands the potential therapeutic use of the Mn porphyrins to disease models that are not SOD based.
Project description:BACKGROUND:Mn/Fe-superoxide dismutase (SOD) is a family of enzymes essential for organisms to be able to cope with oxygen. These enzymes bound to their classical metals catalyze the dismutation of the free radical superoxide anion (O2(-)) to H2O2 and molecular oxygen. E. coli has the manganese-dependent SOD A and the iron-dependent SOD B. METHODS:Strains of E. coli overexpressing SOD A or SOD B were grown in media with different metal compositions. SODs were purified and their metal content and SOD activity were determined. Those proteins were incubated with H2O2 and assayed for oxidation of Amplex red or o-phenylenediamine, consumption of H2O2, release of iron and protein radical formation. Cell survival was determined in bacteria with MnSOD A or FeSOD A after being challenged with H2O2. RESULTS:We show for the first time that the bacterial manganese-dependent SOD A when bound to iron (FeSOD A) has peroxidase activity. The in vivo formation of the peroxidase FeSOD A was increased when media had higher levels of iron because of a decreased manganese metal incorporation. In comparison to bacteria with MnSOD A, cells with FeSOD A had a higher loss of viability when exposed to H2O2. GENERAL SIGNIFICANCE:The biological occurrence of this fundamental antioxidant enzyme in an alternative iron-dependent state represents an important source of free radical formation.
Project description:Human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) undergoes site-specific and random fragmentation by non-enzymic glycosylation (glycation). Released Cu2+ from the glycated Cu,Zn-SOD probably facilitates a Fenton reaction to convert H2O2 into hydroxy radical, which then participates in the non-specific fragmentation [Ookawara et al. (1992) J. Biol. Chem. 267, 18505-18510]. In the present study, we investigated the effects of glycated Cu,Zn-SOD on cloned DNA fragments and nuclear DNA and analysed the formation of 8-hydroxydeoxyguanosine (8-OH-dG). Incubation of cloned DNA fragments with Cu,Zn-SOD and reducing sugars resulted in cleavage of the DNA. The extent of the cleavage corresponded to the reducing capacity of the sugar. Metal-chelating reagents, EDTA and bathocuproine, and an H2O2 scavenger, catalase, inhibited the DNA cleavage. Hydroxy radical scavengers and aminoguanidine, an inhibitor of glycation, also inhibited the reaction. Moreover, the glycation of Cu,Zn-SOD caused the substantial formation of 8-OH-dG in DNA. When isolated nuclei were incubated with CuCl2 plus H2O2, nuclear DNA cleavage was observed. Incubation of isolated nuclei with Cu,Zn-SOD that had been pre-incubated with glucose also resulted in nuclear DNA cleavage. These results suggest that hydroxy radical is produced through a Fenton reaction by Cu2+ and H2O2 released from the glycated Cu,Zn-SOD, and participates in nuclear DNA cleavage. This mechanism may partly explain the deterioration of organs under diabetic conditions.
Project description:A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.
Project description:This study investigates the effect and mechanisms of low pressure dielectric barrier discharge (LPDBD) produced with Ar/O2 and Ar/Air technique causing biological stimulation leading to improved germination and growth in wheat. Both plasma treatments caused rougher and chapped seed surface along with noticeable improvement in seed germination in wheat. Beside this, seed H2O2 concentration significantly increased compared to controls subjected to Ar/O2 and Ar/Air while this phenomenon was more pronounced due to Ar/Air plasma. Analysis of plants grown from the plasma treated seeds showed significant improvement in shoot characteristics, iron concentration, total soluble protein and sugar concentration in comparison with the controls more efficiently due to Ar/O2 plasma than that of Ar/Air. Further, none of the plasma treatments caused membrane damage or cell death in root and shoot of wheat. Interestingly, Ar/O2 treated plants showed a significant increase (2-fold) of H2O2 compared to controls in both root and shoot, while Ar/Air plasma caused no changes in H2O2. This phenomenon was supported by the biochemical and molecular evidence of SOD, APX and CAT in wheat plants. Plants derived from Ar/O2 treated seeds demonstrated a significant increase in SOD activity and TaSOD expression in roots of wheat, while APX and CAT activities along with TaCAT and TaAPX expression showed no significant changes. In contrast, Ar/Air plasma caused a significant increase only in APX activity in the shoot. This suggests that Ar/O2 plasma caused a slight induction in H2O2 accumulation without triggering the H2O2 scavengers (APX and CAT) and thus, efficiency affect growth and development in wheat plants. Further, grafting of control and Ar/O2 treated plants showed a significant increase in shoot biomass and H2O2 concentration in grafts having Ar/O2 rootstock regardless of the type scion attached to it. It indicates that signal driving Ar/O2 plasma mediated growth improvement in wheat is possibly originated in roots. Taken together, this paper delivers new insight into the mechanistic basis for growth improvement by LPDBD technique.
Project description:Superoxide dismutase (SOD, EC 188.8.131.52) is an important metal-containing antioxidant enzyme that provides the first line of defense against toxic superoxide radicals by catalyzing their dismutation to oxygen and hydrogen peroxide. SOD is classified into four metalloprotein isoforms, namely, Cu/Zn SOD, Mn SOD, Ni SOD and Fe SOD. The structural models of soybean SOD isoforms have not yet been solved. In this study, we describe structural models for soybean Cu/Zn SOD, Mn SOD and Fe SOD and provide insights into the molecular function of this metal-binding enzyme in improving tolerance to oxidative stress in plants.