H2O2 generated by alveolar macrophages has been linked to the development pulmonary fibrosis, but little is known about its source, mechanism of production and exact role upon alveolar macrophage activation. In this study, we found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H2O2 and have high expression of Cu,Zn-SOD. Cu,Zn-SOD localized to the mitochondrial intermembrane space (IMS) in asbestosis patients and asbestos induced translocation of Cu,Zn-SOD to the IMS. This process was unique to macrophages and dependent on functional mitochondrial respiration. The presence of at least one of the conserved cysteines was required for disulfide bond formation and mitochondrial translocation. These conserved cysteine residues were also necessary for enzyme activation and H2O2 generation. Cu,Zn-SOD-mediated H2O2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant as Cu,Zn-SOD-/- mice generated significantly less H2O2, had less oxidative stress, and were protected from developing pulmonary fibrosis. This protective mechanism is closely related to the alveolar macrophage activation and polarization in Cu,Zn-SOD-/- mice, as they had a dominant pro-inflammatory phenotype. Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. The pro-inflammatory M1 macrophages have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain settings. We demonstrate that overexpression of Cu,Zn-SOD promoted macrophages polarization into an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis. The mechanism which Cu,Zn-SOD-mediated H2O2 utilizes to modulate macrophage M2 polarization is through redox regulation of a critical cysteine in STAT6. The polarization process, at least partially, was regulated by epigenetic modulation. We show that STAT6 was indispensable for Cu,Zn-SOD-mediated M2 polarization. STAT6 upregulated Jmjd3, a histone H3 lysine 27 demethylase, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD plays a paradoxical role. The study highlights the importance of mitochondrial Cu,Zn-SOD and redox signals in macrophage polarization and fibrosis development. These observations demonstrate that the Cu,Zn-SOD-STAT6-Jmjd3 pathway is a novel regulatory mechanism for M2 polarization and that leflunomide is a potential therapeutic agent in the treatment of pulmonary fibrosis.

<p>Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. </p><p>This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.</p>

Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by the progressive degeneration of motor neurons. Dominantly-inherited mutations to the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) cause 3-6% of all ALS cases. The complete mechanism behind the toxicity of mutant SOD1 remains unclear, although significant evidence points to aberrant or incomplete metal-binding having a role in a toxic gain-of-function. However, the relevance of the metal-binding of SOD1 to mutant-SOD1-linked ALS remains controversial. Direct assessments of protein metal-binding from transgenic, SOD1-overexpressing rodent models of the disease are difficult to acquire due to the non-covalent nature of the interaction. The relatively small amount of disease-afflicted spinal cord tissue in which the motor neurons reside compounds the difficulty of measuring the protein metal binding of SOD1 from transgenic mice. This dissertation addresses the metals bound to SOD1 throughout the disease course in transgenic mice using a novel mass spectrometry assay. The methodology developed here offers the first detailed examination of partially unfolded intermediates of SOD1 present in the spinal cord of pre-symptomatic, symptomatic, and end-stage transgenic mice overexpressing the ALS-associated SOD1 mutation G93A (glycine mutated to alanine at position 93). These results were compared to age-matched transgenic mice expressing wild-type SOD1 that do not develop ALS symptoms. To extract SOD1 from relevant spinal cord tissue, a 300 ��m necropsy punch was used to remove a small piece of tissue from the ventral or dorsal gray matter of a 1 mm-thick slice of spinal cord. Physiological salts that interfere with electrospray mass spectrometry were removed by binding the proteins to a C4 Ziptip��, a pipette tip containing hydrophobic, reversed-phase packing material. Washing the Ziptip-bound proteins with water eliminated interfering salts. Bound proteins could then be eluted into a mass spectrometer with low concentrations of acetonitrile plus formic acid. Electrospray ionization conditions were determined that could keep both copper and zinc bound to SOD1. Using a high-resolution Fourier transform-ion cyclotron resonance mass spectrometer, we used the assay to collect isotopically-resolved protein mass data. Theoretical protein isotope distributions were calculated from the empirical formulas of SOD1 and matched to the experimental data with a least squares fitting algorithm to determine the multiple intermediates of SOD1 present. Spinal cord tissue, wild-type in particular, was notable for containing significantly more one-metal SOD1 than any other tissue, despite having 3-fold less SOD1 than liver. We quantitatively compared the levels of soluble, partially unfolded intermediates of SOD1 from wild-type and G93A SOD1 spinal cords. Wild-type mouse spinal cord contained significantly more of all of the partially unfolded intermediates copper-deficient SOD1, disulfide reduced SOD1, and apo SOD1. The amount of zinc-containing SOD1 was exceptionally high in wild-type mice, comprising 60% of the total SOD1 in wild-type spinal cord. The larger amounts of these SOD1 intermediates in wild-type transgenic mice indicate that they are not directly responsible for toxicity in vivo. However, copper-containing, zinc-deficient SOD1 was the one species found in higher concentrations in G93A SOD1 spinal cord. The concentration was on average 0.6-0.8 ��M in G93A spinal cord, compared to 0.1-0.3 ��M zinc-deficient SOD1 found in the wild-type mouse spinal cord. A concentration above 0.5 ��M zinc-deficient SOD1 was sufficient to induce motor neuron death in vitro. These results suggest that copper-containing, zinc-deficient SOD1 could be the toxic species responsible for motor neuron death in ALS. / Graduation date: 2013

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. / The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.

No description available.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H2O2 treatment and compared to that of FeSOD. It’s found that the responses to H2O2 treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe3+ reduction was not observed in Fe(Mn)SOD even H2O2 is believed to oxidize proteins via highly reactive intermediates including Fe and formed via Fe2+, which is true in FeSOD. What’s more, the activities of Fe(Mn)SOD and FeSOD were totally different in the ABTS assay or Amplex Red assay. These results indicated that the mechanism of peroxidase reaction of Fe(Mn)SOD is not identical to that of FeSOD.

Maintainence of appropriate redox homeostasis is crucial for processes such as protein folding in the endoplasmic reticulum (ER) and to minimise genesis of oxidative stress. Previous studies have indicated a possible link between ER stress and production of reactive oxygen species (ROS) although the cellular mechanisms involved were not fully elucidated. To investigate the cellular mechanisms involved in tolerance to oxidative stress and ER stress, genome-wide screens were performed to identify mutants sensitive to chronic ER stress induced by dithiothreitol and tunicamycin. These screens identified the Cu,Zn superoxide dismutase (SOD1) and genes involved in NADPH generation (RPE1, TKL1) as important for chronic ER stress tolerance. Superoxide anion has been identified as one of the ROS generated during ER stress. The ER oxidoreductase Ero1p, previously implicated in ROS production in vitro, did not appear to be a source of superoxide when the protein was over-expressed. It was also found that cellular NADP(H) levels affected induction of the unfolded protein response (UPR), since cells lacking TKL1 or RPE1 exhibited decreased UPR induction. These data indicate an important role for superoxide dismutase and cellular NADP(H) in survival of cells during ER stress. Subsequent analysis determined that NADPH generation was also required for adaptation to H2O2. Mutants affected in NADPH production were chronically sensitive to H2O2 but resistant to an acute dose. These mutants over-accumulated reduced glutathione (GSH) but maintained normal cellular redox homeostasis. This over- production of GSH was not regulated at the transcriptional level of GSH1 encoding ??- glutamyl cysteine synthetase. These data raise the important question as to how cells maintain cellular glutathione redox balance. To better understand how cells respond to perturbations in glutathione redox homeostasis, cells deleted for GLR1, encoding GSSG reductase, were exposed to extracellular oxidised glutathione (GSSG) and intracellular GSH and GSSG were monitored over time. Intriguingly cells lacking GLR1 showed increased levels of GSH accumulation upon GSSG treatment in a manner independent of GSH synthesis. It was subsequently found that the cytosolic thioredoxin-thioredoxin reductase system contributes to the reduction of GSSG in vivo.

Made available in DSpace on 2014-06-11T19:23:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-22Bitstream added on 2014-06-13T20:10:55Z : No. of bitstreams: 1 corniani_n_me_botfca.pdf: 1146521 bytes, checksum: fa6a3da1e11828289d5c9cb1e7208258 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As espécies do gênero Eucalyptus apresentam notável capacidade de extração de água e nutrientes, assimilação fotossintética e crescimento, permitindo seu cultivo em ambientes considerados impróprios para muitas outras espécies. Além dos fatores ambientais atuando adversamente no desempenho das plantas, atualmente estas estão sujeitas aos efeitos das mudanças ambientais globais causadas pelas atividades antrópicas, tais como o aumento na incidência da radiação ultravioleta-B (RUV-B) em conseqüência da destruição da camada de ozônio. Estudos recentes têm mostrado que a RUV-B promove aumento no nível de espécies reativas de oxigênio (ERO), ocasionando estresse oxidativo nas plantas. Entretanto, as plantas apresentam um sistema de defesa contra o estresse oxidativo, constituído de enzimas antioxidantes, tais como superóxido dismutase (SOD, EC 1.15.1.1) e peroxidase (POD, EC 1.11.1.7), além de outros compostos, como antocianinas, carotenóides e compostos fenólicos, que impedem a formação de ERO ou eliminam as já existentes. Há algum tempo, tem sido verificada em plantas a presença de óxido nítrico (NO), radical livre endógeno que possui a capacidade de controlar o nível e a toxicidade das ERO. Portanto, o objetivo deste trabalho foi investigar o possível efeito citoprotetor do NO em plantas de eucalipto (Eucalyptus urograndis) expostas à RUV-B. Assim, foi realizado um primeiro experimento no qual plantas de eucalipto foram expostas à RUV-B em baixa (controle) e elevada incidência, com o intuito de verificar se a radiação promove estresse oxidativo nesta espécie. Foram realizadas coletas de lâminas foliares aos cinco, dez e quinze dias após o início da exposição à RUV-B para posterior determinação do nível de peroxidação lipídica. A exposição por 15 dias à RUV-B... / The species of the genus Eucalyptus present notable capacity to extract water and nutrients, photosynthetic assimilation and growth, allowing cultivation in ambient considered inappropriate for many other species. Besides the environmental factors acting adversely in the performance of the plants, nowadays they are subject to the effects of the global environmental changes caused by human activities, such as the increase in the incidence of the ultraviolet-B radiation (UV-B) in consequence of the ozone layer destruction. Recent studies show that UV- B radiation promotes increase in the level of reactive oxygen species (ROS), causing oxidative stress in plants. However, plants have a defence system against oxidative stress, constituted of antioxidant enzymes, such as, superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), and others compounds, such as anthocyanins, carotenoids and phenolic compounds, that avoid ROS formation or eliminate the already existent. There has been long, it was noticed the presence of nitric oxide (NO) in plants, endogenous free radical able to control the ROS level and toxicity. Therefore, the aim of this work was to investigate the possible citoprotector effect of the NO in eucalyptus (Eucalyptus urograndis) plants subjected to UV-B radiation. So, a first experiment was setup in which plants of eucalyptus were subjected to radiation in low (control) and high incidence of UV-B, to verify if UV-B radiation promotes oxidative stress in this specie. Leaves were collected at five, ten and fifteen days after the beginning of exposure to UV-B for subsequent evaluation of the level of lipid peroxidation. Exposure for 15 days to RUV-B increased the lipoperoxide content in the eucalyptus... (Complete abstract click electronic access below)