Spelling suggestions: "subject:"thioredoxin"" "subject:"thioredoxins""
111 |
ROLE OF SULFIREDOXIN INTERACTING PROTEINS IN LUNG CANCER DEVELOPMENTChawsheen, Hedy 01 January 2016 (has links)
Sulfiredoxin (Srx) is an antioxidant enzyme that can be induced by oxidative stress. It promotes oncogenic phenotypes of cell proliferation, colony formation, migration, and metastasis in lung, skin and colon cancers. Srx reduces the overoxidation of 2-cysteine peroxiredoxins in cells, in addition to its role of removing glutathione modification from several proteins. In this study, I explored additional physiological functions of Srx in lung cancer through studying its interacting proteins. Protein disulfide isomerase (PDI) family members, thioredoxin domain containing protein 5 (TXNDC5) and protein disulfide isomerase family A member 6 (PDIA6), were detected to interact with Srx. Therefore, I proposed that TXNDC5 and PDIA6 are important for the oncogenic phenotypes of Srx in lung cancer.
In chapter one, I presented background information about the role of Srx as an antioxidant enzyme in cancer. I also explained the functional significance of PDIs as oxidoreductase and chaperones in cells. In chapter two, I verified the Srx-TXNDC5/PDIA6 interaction in HEK293T and A549 cells by co-immunoprecipitation and other assays. In TXNDC5 and PDIA6, the N-terminal thioredoxin-like domain (D1) is determined to be the main platform for interaction with Srx. The Srx-TXNDC5 interaction was enhanced by H2O2 treatment in A549 cells. Srx was determined to localize in the endoplasmic reticulum (ER) of A549 cells along with TXNDC5 and PDIA6. This localization was confirmed by both subcellular fractionation and immunofluorescence imaging experiments. In chapter three I focused on studying the physiological function of Srx interacting proteins in the ER. A549 subcellular fractionation results showed that TXNDC5 facilitates Srx retention in the ER. Moreover, TXNDC5 and Srx were found to participate in chaperone activities in lung cancer. Both proteins contributed in the refolding of heat-shock induced protein aggregates. In addition, TXNDC5 and PDIA6 were found to enhance the protein refolding in response to H2O2 treatment. Conversely, Srx appeared to have an inhibitory effect on protein folding under same treatment conditions. Downregulation of Srx, TXNDC5, or PDIA6 significantly reduced cell viability in response to tunicamycin treatment. TXNDC5 knockdown decreased the time required for the splicing of X-box binding protein-1 (XBP-1). In either knockdown Srx or TXNDC5 cells, there was an observable decrease in the expression of GRP78 and the splicing of spliced XBP-1. These results suggest a possible role of Srx in unfolded protein response signaling. TXNDC5 and PDIA6, similar to Srx, contribute to the proliferation, anchorage independent colony formation and migration of lung cancer cells.
In this dissertation I concluded that Srx TXNDC5, and PDIA6 proteins participate in oxidative protein folding in lung cancer. Srx and TXNDC5 can modulate unfolded protein response (UPR) sensor activation and growth inhibition. Furthermore, TXNDC5 and PDIA6 can promote tumorigenesis of lung cancer cells. Therefore, the molecular interaction of Srx with TXNDC5/PDIA6 has the potential to be used as novel therapeutic targets for lung cancer treatment.
|
112 |
Mécanismes cellulaires et moléculaires régissant le métabolisme des semences de céréales : rôle du réseau rédoxines-système antioxydant dans la prédiction de la qualité germinative / Cellular and molecular mechanisms governing the metabolism of the cereals seeds : role of the network antioxidant system/redoxins in the prediction of the germinative quality.Zahid, Abderrakib 16 July 2010 (has links)
Une meilleure compréhension de la physiologie de la semence des céréales constitue certainement un moyen pour améliorer et développer de nouvelles variétés capables de correspondre aux besoins économiques et écologiques du moment. Les rédoxines constituent des marqueurs intéressants pour appréhender la qualité technologique et germinative du grain de blé en particulier. Le criblage des banques de données a permis d’isoler des isoformes de ces rédoxines. Cette étude a confirmé l’implication des thiorédoxines dans la réduction des protéines de réserve du blé et de maïs. Elle a permis de mettre en évidence un autre rôle de certains isoformes de thiorédoxines h dans la formation de polymères de hauts poids moléculaires. L'inhibition de l’expression de gènes par interférence ADN montre que les thiorédoxines et les glutarédoxines sont impliquées dans la protection contre le stress oxydatif chez le blé. De même, l'application d’un stress biotique simulé par la laminarine a permis de discriminer l'implication de différents marqueurs du stress, et de montrer en particulier que la 1-Cys-Prx peut être considérée comme un indicateur de l'état redox du grain pendant la germination. La mise en place d’une méthode simple et efficace de transformation des céréales via Agrobacterium, constitue un moyen pour comprendre davantage le rôle de ces rédoxines dans la gestion des stress, et les éventuelles conséquences sur la qualité technologique du grain. / A better understanding of the physiology of seed cereal constitutes certainly a means to improve and develop new varieties capable of corresponding to the actual economic and ecological needs. Redoxins are interesting markers to apprehend the technological and germinative quality of wheat seed in particular. The screening of data banks allowed isolating isoforms of these redoxins. This study confirmed the implication of thioredoxins in the reduction of storage proteins in wheat and corn seeds. It allowed to bring to light another role of some thioredoxins h isoforms in the formation of high molecular weights polymers. The inhibition of the expression of genes by DNA interference shows that thioredoxins and glutaredoxins are involved in the protection against oxidative stress in wheat. Also, the application of a biotic stress simulated by laminarin allowed to discriminate the implication of various stress markers, and to highlight in particular that the 1-Cys-Prx can be considered as an indicator of the redox state of the grain during germination and seedling. The implementation of a simple and effective method of transformation of cereal via Agrobacterium constitutes a means to understand more on the role of these redoxins in the management of the stress, and the possible consequences on the technological quality of the seed.
|
113 |
Polimorfismos nos genes que codificam a glutationa peroxidase-4, a tiorredoxina e a proteína de interação com a tiorredoxina modulam a susceptibilidade à doença renal em portadores de diabetes mellitus tipo 1 / Polymorphisms in the genes coding for glutathione peroxidase-4, thioredoxin and thioredoxin interaction protein modulate the risk for renal disease in type 1 diabetes patientsMonteiro, Maria Beatriz Camargo de Almeida 14 March 2012 (has links)
INTRODUÇÃO: evidências sugerem a participação de fatores genéticos na susceptibilidade para o desenvolvimento das complicações renais em pacientes portadores de diabetes mellitus tipo 1 (DM1). Vários genes relacionados às vias bioquímicas induzidas pela hiperglicemia têm sido investigados e o estresse oxidativo foi reconhecido como o principal mecanismo patogênico responsável pelo dano celular causado pela hiperglicemia no DM. Assim, genes que codificam enzimas que participam de vias antioxidantes endógenas são candidatos a conferirem susceptibilidade, ou proteção, contra as complicações renais. Os sistemas da glutationa, glutarredoxina, tiorredoxina e a enzima transcetolase são importantes mecanismos de defesa celular contra o estresse oxidativo. OBJETIVOS: avaliar a associação entre os seguintes polimorfismos de um único nucleotídeo (SNP) e a doença renal em pacientes diabéticos tipo 1: -2030 T/G (rs34071297) e +718C/T (rs713041) no gene que codifica a glutationa peroxidase 4 (GPX4); -3310 G/C (rs10427424) no gene que codifica a glutationa sintetase (GSS); -247 A/G (rs2978668) no gene que codifica a glutationa redutase (GSR); -2763 A/G (rs6556885) no gene que codifica a glutarredoxina (GLRX); -224 T/A (rs2301242) no gene que codifica a tiorredoxina (TXN); +402 T/C (rs7211) no gene que codifica a proteína de interação com a tiorredoxina (TXNIP); -192 G/A (rs3788319) no gene que codifica a tiorredoxina redutase 2 (TXNRD2) e -3787 T/G (rs7637934) e -1410 T/C (rs11130365) no gene que codifica a transcetolase (TKT). CASUÍSTICA E MÉTODOS: 443 pacientes (192 do sexo masculino e 251 do sexo feminino) com DM1 com mais de 10 anos de diagnóstico foram classificados conforme a presença ou ausência das seguintes complicações: (1) nefropatia diabética franca (ND), definida por macroalbuminúria a proteinúria persistente; (2) nefropatia diabética estabelecida (NDE), definida por macroalbuminúria a proteinúria persistente ou RFGe < 60 mL/min/1,73 m2 ou pacientes em terapia de substituição renal e (3) ritmo de filtração glomerular estimado (RFGe) ou < que 60 mL/min/1,73 m2. O teste de Pearson 2 foi usado para comparar as frequências dos genótipos e a magnitude de associação foi estimada pelo cálculo do odds ratios (OR). A OR ajustada foi estimada por regressão logística para possíveis fatores de confusão (sexo, idade ao diagnóstico, tempo de diabetes, HbA1c, concentrações plasmáticas de colesterol e triglicérides e presença de hipertensão arterial). Pacientes controle não diabéticos também foram incluídos para avaliar se os SNPs não confeririam susceptibilidade para o DM1. RESULTADOS: A presença de pelo menos um alelo T do polimorfismo +718C/T no gene GPX4 conferiu proteção para a presença de ND estabelecida (OR=0,41; IC 95% 0,19-0,83, p= 0,0146) e ND franca (OR=0,37; IC 95% 0,15- 0,85; p= 0,021) na população masculina mesmo após ajuste para os fatores de confusão e a presença de dois alelos polimórficos A no polimorfismo -224 T/A no gene TXN conferiu risco para a presença de ND franca na população feminina após ajuste para os fatores de confusão (OR= 4,06; IC 95% 1,59-10,6, p= 0,0035). O genótipo TT para o SNP +402 T/C do gene da TXNIP foi mais frequente nos pacientes portadores de DM1 em relação aos controles não diabéticos. O genótipo CC do SNP TXNIP +402 T/C conferiu proteção para a presença de ND estabelecida em homens mesmo após ajuste para os fatores de confusão (OR=0,45; IC 95% 0,22-0,91; p= 0,02). CONCLUSÕES: Os SNPs +718C/T (rs713041) no gene GPX4, -224 T/A (rs2301242) no gene TXN e +402 T/C (rs7211) no gene TXNIP, modulam o risco para o comprometimento renal na população de portadores de DM1 estudada / INTRODUCTION: there is evidence suggesting that genetic factors are involved in the susceptibility to the development of renal complications in patients with type 1 diabetes mellitus (DM1). Several genes related to the mechanisms of hyperglycemia-induced cell damage have been investigated. Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. Thus, genes that encode enzymes involved in endogenous antioxidant pathways may be candidates for conferring risk or protection against renal complications. The glutathione, glutaredoxin, and thioredoxin systems and transketolase enzyme are important mechanisms of cellular defense against oxidative stress. OBJECTIVES: to evaluate the association between the following single nucleotide polymorphisms (SNPs) and renal disease in type 1 diabetic patients: -2030 T/G (rs34071297) and +718C/T (rs713041) in the gene encoding glutathione peroxidase 4 (GPX4); -3310 G/C (rs10427424) in the gene encoding glutathione synthetase (GSS); -247 A/G (rs2978668) in the gene encoding glutathione reductase (GSR); -2763 A/G (rs6556885) in the gene encoding glutaredoxin (GLRX); -224 T/A (rs2301242) in the gene encoding thioredoxin (TXN); +402 T/C (rs7211) in gene encoding thioredoxin interacting protein (TXNIP); -192 G/A (rs3788319) in the gene encoding thioredoxin reductase 2 (TXNRD2); - 3787 T/G (rs7637934) and -1410 T/C (rs11130365) in the gene encoding transketolase (TKT). MATERIALS AND METHODS: 443 patients (192 males and 251 females) with type 1 diabetes duration > 10 years were grouped according to presence or absence of the following complications: (1) overt diabetic nephropathy (DN) defined by persistent macroalbuminuria to proteinuria; (2) established diabetic nephropathy (EDN), defined by persistent macroalbuminuria or proteinuria or estimated glomerular filtration rate (RFGe) < 60 mL/min/1.73 m2 or patients under renal replacement therapy and (3) RFGe or < 60 mL/min/1.73 m2. Pearsons 2 test was performed to compare the genotype frequencies and magnitude of association was estimated using odds ratios (OR). Adjusted OR was estimated by logistic regression for possible confounders (sex, age at diagnosis, diabetes duration, HbA1C, cholesterol and triglyceride concentrations and the presence of hypertension). Nondiabetic subjects were also included. RESULTS: The presence of at least one T polymorphic allele of the SNP GPX4 +718 C/T was protective against EDN (OR = 0.41, CI 95% 0.19- 0.83, p= 0.0146) and against overt DN (OR=0.37; IC 95% 0.15-0.85; p= 0.021) in the male population even after adjustment for possible confounders. The presence of two polymorphic alleles of the SNP TXN -224 T/A conferred independent risk for the presence of overt DN in the female population after adjustment for possible confounders (OR = 4.06, CI 95% 1.59- 10.6, p= 0.0035). The TT genotype for the SNP TXNIP +402 T/C was more frequent in patients with type 1 diabetes compared to nondiabetic controls. The genotype CC of the SNP TXNIP +402 T/C was protective against EDN in male population even after adjustment for possible confounders (OR=0.45; IC 95% 0.22-0.91; p= 0.02) CONCLUSIONS: The SNPs GPX4 +718C/T (rs713041), TXN -224 T/A (rs2301242) and TXNIP +402T/C (rs7211) modulate the risk for renal disease in the studied population of type 1 diabetes patients
|
114 |
Implication du système Thiorédoxine des chondrocytes humains soumis à un stress glucosé, en hypoxie et en normoxie : effets du Resvératrol / Implication of thioredoxin system in human chondrocytes subjected to high glucose stress, under hypoxia and normoxia : effects of ResveratrolLe Clanche, Solenn 06 July 2015 (has links)
L’arthrose est une maladie dégénérative de l’articulation caractérisée par une dégradation du cartilage, une inflammation de la membrane synoviale et un remodelage de l’os sous-chondral. En conditions physiologiques, les chondrocytes, seul type cellulaire du cartilage, sont en hypoxie (≈ 2% d’oxygène). Le cartilage étant un tissu avascularisé, il existe un gradient de concentration en oxygène au sein des différentes couches du cartilage. Lors du développement de l’arthrose, la dégradation du cartilage provoque une rupture de ce gradient, exposant ainsi les cellules des couches profondes à des concentrations en oxygène beaucoup plus élevées et induisant des modifications de leur métabolisme, ce qui induit leur dysfonction. Le syndrome métabolique est défini par un ensemble de perturbations glucidiques, lipidiques et vasculaires menant au développement de maladies cardiovasculaires et au diabète de type 2. Récemment, un lien entre arthrose et syndrome métabolique a été suggéré, introduisant une notion d’arthrose métabolique. Au cours de cette étude, nous nous sommes intéressés au lien entre arthrose, syndrome métabolique et stress oxydant induit par de fortes concentrations de glucose. Dans la première partie de ce travail, nous avons étudié les effets in vitro de 25 mM de glucose sur une lignée de chondrocytes humains immortalisés (T/C28a2), en hypoxie (2% d’oxygène) et en normoxie (21% d’oxygène). Nous avons montré que le glucose à 25 mM induisait la production d’espèces réactives de l’oxygène (ERO) et de l’azote, l’activation de la caspase 3, la production d’interleukine 6 (IL-6), la diminution de l’activité lysyl oxydase (LOX), qui est impliquée dans les liaisons de pontage des fibres de collagène et d’élastine, ainsi que l’activation du système thiorédoxine (Trx). Ce dernier est un système de défense anti-oxydant endogène composé de la thiorédoxine, de la thiorédoxine réductase (TR) et de Txnip, qui intervient dans le maintien de l’homéostasie cellulaire en réduisant les protéines oxydées, contrôlant ainsi l’environnement redox des cellules. Les effets du glucose 25 mM ont été observés dans les deux conditions d’oxygène étudiées, cependant la réponse cellulaire en normoxie était plus précoce qu’en hypoxie. Nous avons également pu mettre en évidence un rôle de régulateur négatif de la Trx-1 sur la production d’IL-6 faisant intervenir la voie de signalisation p38MAPK. Dans la deuxième partie de ce travail, nous nous sommes intéressés aux effets de l’apport exogène de resvératrol sur les modifications induites par le glucose à 25 mM. Le resvératrol (3,4’,5-trihydroxystilbène) est un polyphénol de la famille des stilbènes, connu pour ses multiples propriétés anti-oxydantes, anti-inflammatoires, anti-diabétiques et anti-cancer. Nous avons pu observer que le resvératrol à 25 μM était capable de diminuer les effets délétères provoqués par le glucose à 25 mM. Cependant, la biodisponibilité du resvératrol est très limitée, empêchant son utilisation en thérapeutique humaine. Par conséquent, dans la troisième partie de cette étude, nous nous sommes intéressés au développement de nouvelles formulations galéniques de resvératrol (nano-émulsions (NE)) et à leurs effets sur un modèle de cellules endothéliales aortiques bovines (BAEC), sur les T/C28a2 ainsi que sur des chondrocytes humains en culture primaire provenant de cartilage de patients arthrosiques. Nous avons montré qu’une des NE permettait d’augmenter le passage intracellulaire de resvératrol dans les deux modèles étudiés et d’en potentialiser les effets protecteurs contre un stress oxydant. Cette NE s’est également montrée efficace dans le rétablissement de l’activité LOX dans les cellules de patients arthrosiques. En conclusion, nous avons montré que le glucose à 25 mM avait des effets délétères sur les chondrocytes de la lignée T/C28a2 et que l’apport exogène de resvératrol permettait de lutter contre ses effets. (...) / Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, inflammation of synovial membrane and subchondral bone remodelling. Under physiological conditions, chondrocytes - the only cell type found in cartilage - are under hypoxia (around 2% of oxygen). As cartilage is an avascular tissue, an oxygen gradient is established from the superficial to the deeper layers. During OA development, cartilage degradation is responsible for a break in this gradient; consequently, cells from the deepest layers are exposed to higher oxygen concentrations inducing modifications in cell metabolism leading to their dysfunction. Metabolic syndrome (MetS) is defined by a cluster of factors (impairment of glucose and lipid metabolism, vascular dysfunctions…) leading to cardiovascular diseases and type 2 diabetes development. Recently, a link between OA and MetS has been suggested, introducing a notion of metabolic OA. We have focused our study on the link between OA, MetS and oxidative stress induced by high glucose concentrations. In the first part of this study, we have determined the in vitro effects of 25 mM glucose on an immortalized human chondrocyte cell line (T/C28a2), under hypoxia (2% oxygen) and normoxia (21% oxygen). We demonstrated that 25 mM glucose induced radical oxygen species (ROS) and nitric oxide production, caspase 3 activation, interleukin 6 (IL-6) production, decrease in lysyl oxidase (LOX) activity (involved in type II collagen crosslinks), and activation of the thioredoxin (Trx) system. Trx system is an endogenous anti-oxidant system, composed by thioredoxin, thioredoxin reductase (TR) and Txnip; it is involved in cellular homeostasis by reducing oxidized proteins, thereby controlling cellular redox environment. Effects of 25 mM glucose have been observed under both oxygen conditions; nevertheless, cellular response under normoxia underwent earlier than under hypoxia. We have also highlighted Trx-1 as a negative regulator of IL-6 production through p38MAPK signalling pathway. In the second part of this study, we have focused our work on the effects of the addition of an exogenous antioxidant, i.e. resveratrol, on the modifications induced by 25 mM glucose. Indeed, resveratrol (3,4’,5-trihydroxystilbene) is a polyphenol of the stilbene family, known for its multiple anti-inflammatory, anti-oxidative, anti-diabetes and anti-cancer properties. We have observed that 25 μM resveratrol was able to decrease deleterious effects induced by 25 mM glucose. However, resveratrol bioavailability is very low, avoiding its use in human therapeutic strategy. Consequently, in the third part of this study, we have developed new galenic formulations of resveratrol, i.e. nano-emulsions (NEs) and determined their effects on a bovine aortic endothelial cells (BAEC) model, on T/C28a2 cells and also on primary cultures of human chondrocytes from osteoarthritic cartilages. One of our NEs was able to increase resveratrol intracellular passage in both cellular models, and to increase the protective effects of resveratrol against oxidative stress. This NE was also efficient in the normalization of LOX activity in osteoarthritic chondrocytes. To conclude, we have demonstrated that 25 mM glucose induced deleterious effects on chondrocytes of the T/C28a2 cell line, and that an exogenous supply in resveratrol allowed to counteract these effects. Development of a new galenic formulation of resveratrol opens new interesting prospects in human therapeutic strategy against OA associated with MetS.
|
115 |
Efeito pró-oxidante do hidroperóxido de urato sobre proteínas sensíveis às alterações redox: implicações na resposta inflamatória / Pro-oxidant effect of urate hydroperoxide on redoxsensitive proteins: implications on inflammatory reponseCarvalho, Larissa Anastácio da Costa 19 May 2017 (has links)
O hidroperóxido de urato (HOOU) é o produto da oxidação do ácido úrico por peroxidases. Sua produção é favorecida durante a inflamação e hiperuricemia, uma vez que há grande quantidade de ácido úrico, peroxidases inflamatórias e superóxido. Neste sentido, o objetivo deste estudo foi avaliar o efeito do hidroperóxido de urato sobre proteínas sensíveis à modulação redox em um ambiente inflamatório asséptico e outro que imita infecção. Assim, nesta tese comparou-se a estrutura química do HOOU obtido fotoquimicamente daquele obtido através da catálise enzimática pela mieloperoxidase. A obtenção do HOOU por foto-oxidação permitiu o melhor isolamento do composto. Este oxidante foi capaz de reagir especificamente com os aminoácidos contendo enxofre (metionina e cisteína). Neste sentido, foi investigada sua reatividade com tiol-peroxidases detoxificadoras de peróxido, a peroxiredoxina 1 e 2 (Prx1 e Prx2). O HOOU apresentou cinética rápida de reação com a Prx1, k = 4,9 × 105 M-1s-1 e Prx2, k = 2,3 × 106 M-1s-1, o que as torna um provável alvo celular, além disso, foi capaz de oxidar a Prx2 de eritrócitos humanos, mostrando ser capaz de atravessar a membrana plasmática. Além das Prxs, a albumina do soro também desempenha papel importante na homeostase redox. O HOOU foi capaz de oxidar a albumina com constante de velocidade de 0,2 × 102 M-1s- 1. Outra tiol-proteína com importante função na homeostase e sinalização redox é a tioredoxina (Trx). A Trx foi oxidada pelo HOOU com constante de reação de 2,8 × 102 M-1s-1 e foi liberada juntamente com a Prx1 e Prx2 das células de macrófagos humanos (linhagem THP-1) quando estas células foram incubadas com HOOU. A liberação dessas proteínas é reconhecidamente um sinal de estresse celular. Assim o HOOU pode estar envolvido na exacerbação do estresse oxidativo em ambiente inflamatório. Quando neutrófilos (linhagem HL- 60) e macrófagos humanos (linhagem THP-1) foram incubados na presença de ácido úrico e Pseudomonas aeruginosa houve uma diminuição na produção de ácido hipocloroso (HOCl). Isto se deveu à competição entre ácido úrico e cloreto pela mieloperoxidase e resultou em menor atividade microbicida pelas células, demonstrando que a formação do HOOU não contribui e, ao contrário, prejudica a atividade microbicida das células inflamatórias. Dessa forma, a oxidação do ácido úrico e formação do hidroperóxido de urato tanto altera a atividade microbicida das células inflamtárias, quanto leva à oxidação de tiósproteínas importantes para manutenção da homeostase redox. Assim, o HOOU pode ser o responsável pelos efeitos pró-oxidantes e pró-inflamatórios do ácido úrico solúvel, e isso indica que o papel antioxidante do ácido úrico deve ser revisto em situações de inflamação. / Urate hydroperoxide (HOOU) is the product of the oxidation of uric acid by peroxidases. The formation of HOOU is favored during inflammation and in hyperuricemia, where there is plenty amount of uric acid, inflammatory peroxidases and superoxide. Therefore, the aim of the present study was to evaluate the effect of urate hydroperoxide on redox sensitive proteins in an inflammatory environment and another that mimics infection. In this thesis the chemical structure of the HOOU produced by photo-oxidation was compared to that obtained by myeloperoxidase catalysis. The chemical production of HOOU allowed a better purification of the compound. This oxidant was able to specifically react with sulfur containing amino acids (methionine and cysteine). In this sense, its reactivity with peroxiredoxins (Prx1 and Prx2) was investigated. HOOU reacted fast with Prx1 k = 4.9 × 105 M-1s-1 and Prx2 k = 2.3 × 106 M-1s-1. In addition, HOOU was able to oxidize Prx2 from intact erythrocytes at the same extend as does hydrogen peroxide. Albumin is an important thiol-containing protein to redox homeostasis in plasma. HOOU was able to oxidize albumin with a rate constant of 0.2 × 102 M-1s-1. Another protein with important function in redox homeostasis is thioredoxin (Trx). Trx was oxidized by HOOU with a rate constant of 2.8 × 102 M-1s-1 and was released together with Prx1 and Prx2 from human macrophages cells (THP-1 cell line) that were incubated with HOOU. The release of these proteins is a signal of cellular stress. Thus, HOOU may be involved in the exacerbation of oxidative stress in inflammatory environments. When neutrophil (HL-60 cell line) and macrophages (THP-1 cell line) were incubated with uric acid and Pseudomonas aeruginosa there was a decrease in hypochlorous acid (HOCl) production because of the competition between chloride and uric acid by myeloperoxidase. It decreased HOCl and impaired the microbicidal activity of the cells, showing that HOOU does not contribute in bacteria clearance. Therefore, the oxidation of uric acid to urate hydroperoxide impairs microbicidal activity and oxidizes thiol-proteins in inflammatory cells contributing to a pro-oxidant status. In this context, the antioxidant role of uric acid in inflammatory response should be reviwed.
|
116 |
Etude de la réponse au stress oxydatif de Scedosporium apiospermum, un champignon filamenteux associé à la mucoviscidose / Oxidative stress response of Scedosporium apiospermum, a filamentous fungus associated with cystic fibrosisStaerck, Cindy 13 December 2017 (has links)
La mucoviscidose est la maladie génétique la plus fréquente dans la population caucasienne. Le genre Scedosporium se situe au deuxième rang parmi les champignons filamenteux isolés des expectorations dans ce contexte. Au niveau pulmonaire, les colonisations/infections entraînent le recrutement de phagocytes qui induisent un stress oxydatif normalement délétère pour les pathogènes. Pour se défendre, ceux-ci ont développé des systèmes antioxydants, notamment diverses enzymes. Ce travail de thèse visait à étudier la réponse au stress oxydatif chez Scedosporium. Tout d’abord, la capacité à germer en présence d’oxydants a été évaluée. Par la suite, trente-trois gènes potentiellement impliqués dans la défense contre le stress oxydatif ont été identifiés. Leur expression en présence d’oxydants et en co-cultures avec des phagocytes suggère un rôle majeur, notamment pour une catalase, une peroxyrédoxine et deux thiorédoxine réductases. Par ailleurs, un mutant défectif pour un gène codant une superoxyde dismutase (SOD) pariétale et spécifique des spores a été produit. L’auranofin, un inhibiteur des thiorédoxine réductases, présente une activité vis-à-vis des Scedosporium et un effet additif avec des triazolés. Un test ELISA a été développé pour le sérodiagnostic des scédosporioses, utilisant une catalase et une Cu/Zn-SOD recombinantes. Ce test sensible et spécifique permet de distinguer les infections à Scedosporium de celles à Aspergillus fumigatus et des colonisations à Scedosporium. Au final, ces résultats indiquent un rôle majeur des enzymes antioxydantes chez Scedosporium, qui pourraient être de véritables facteurs de virulence et donc de nouvelles cibles thérapeutiques. / Cystic fibrosis (CF) is the most common genetic disease in Caucasian populations. The Scedosporium genus ranks the second among the filamentous fungi colonizing the airways of CF patients. In the respiratory tract, colonizations/infections lead to the recruitment of phagocytes which produce an oxidative stress, usually deleterious for pathogens. To defend themselves, pathogens have developed protective antioxidant systems, especially various enzymes. This thesis aimed to study the oxidative stress response in Scedosporium species. First, capacity of several Scedosporium isolates to germinate upon oxidative stress conditions was evaluated. Then, thirty-three genes potentially involved in protection against the oxidative stress were identified. Their overexpression in response to oxidants and in co-cultures with phagocytes suggested a crucial role, especially for one catalase, one peroxiredoxin and the two thioredoxin reductases. A mutant defective for the gene encoding a superoxide dismutase (SOD) anchored to the cell wall and specific for the conidia was produced. Auranofin, a thioredoxin reductase inhibitor, exhibits little anti-Scedosporium activity and an additive effect with triazole drugs. An ELISA was developed for serodiagnosis of scedosporiosis, using recombinant proteins derived from one catalase and a Cu/Zn-SOD. This sensitive and specific assay allows to differentiate Scedosporium infections from Aspergillus fumigatus infections and Scedosporium colonizations. Finally, these results indicate a crucial role of antioxidant enzymes in Scedosporium species, which could therefore be considered as virulence factors and as possible new therapeutic targets.
|
117 |
Efeito pró-oxidante do hidroperóxido de urato sobre proteínas sensíveis às alterações redox: implicações na resposta inflamatória / Pro-oxidant effect of urate hydroperoxide on redoxsensitive proteins: implications on inflammatory reponseLarissa Anastácio da Costa Carvalho 19 May 2017 (has links)
O hidroperóxido de urato (HOOU) é o produto da oxidação do ácido úrico por peroxidases. Sua produção é favorecida durante a inflamação e hiperuricemia, uma vez que há grande quantidade de ácido úrico, peroxidases inflamatórias e superóxido. Neste sentido, o objetivo deste estudo foi avaliar o efeito do hidroperóxido de urato sobre proteínas sensíveis à modulação redox em um ambiente inflamatório asséptico e outro que imita infecção. Assim, nesta tese comparou-se a estrutura química do HOOU obtido fotoquimicamente daquele obtido através da catálise enzimática pela mieloperoxidase. A obtenção do HOOU por foto-oxidação permitiu o melhor isolamento do composto. Este oxidante foi capaz de reagir especificamente com os aminoácidos contendo enxofre (metionina e cisteína). Neste sentido, foi investigada sua reatividade com tiol-peroxidases detoxificadoras de peróxido, a peroxiredoxina 1 e 2 (Prx1 e Prx2). O HOOU apresentou cinética rápida de reação com a Prx1, k = 4,9 × 105 M-1s-1 e Prx2, k = 2,3 × 106 M-1s-1, o que as torna um provável alvo celular, além disso, foi capaz de oxidar a Prx2 de eritrócitos humanos, mostrando ser capaz de atravessar a membrana plasmática. Além das Prxs, a albumina do soro também desempenha papel importante na homeostase redox. O HOOU foi capaz de oxidar a albumina com constante de velocidade de 0,2 × 102 M-1s- 1. Outra tiol-proteína com importante função na homeostase e sinalização redox é a tioredoxina (Trx). A Trx foi oxidada pelo HOOU com constante de reação de 2,8 × 102 M-1s-1 e foi liberada juntamente com a Prx1 e Prx2 das células de macrófagos humanos (linhagem THP-1) quando estas células foram incubadas com HOOU. A liberação dessas proteínas é reconhecidamente um sinal de estresse celular. Assim o HOOU pode estar envolvido na exacerbação do estresse oxidativo em ambiente inflamatório. Quando neutrófilos (linhagem HL- 60) e macrófagos humanos (linhagem THP-1) foram incubados na presença de ácido úrico e Pseudomonas aeruginosa houve uma diminuição na produção de ácido hipocloroso (HOCl). Isto se deveu à competição entre ácido úrico e cloreto pela mieloperoxidase e resultou em menor atividade microbicida pelas células, demonstrando que a formação do HOOU não contribui e, ao contrário, prejudica a atividade microbicida das células inflamatórias. Dessa forma, a oxidação do ácido úrico e formação do hidroperóxido de urato tanto altera a atividade microbicida das células inflamtárias, quanto leva à oxidação de tiósproteínas importantes para manutenção da homeostase redox. Assim, o HOOU pode ser o responsável pelos efeitos pró-oxidantes e pró-inflamatórios do ácido úrico solúvel, e isso indica que o papel antioxidante do ácido úrico deve ser revisto em situações de inflamação. / Urate hydroperoxide (HOOU) is the product of the oxidation of uric acid by peroxidases. The formation of HOOU is favored during inflammation and in hyperuricemia, where there is plenty amount of uric acid, inflammatory peroxidases and superoxide. Therefore, the aim of the present study was to evaluate the effect of urate hydroperoxide on redox sensitive proteins in an inflammatory environment and another that mimics infection. In this thesis the chemical structure of the HOOU produced by photo-oxidation was compared to that obtained by myeloperoxidase catalysis. The chemical production of HOOU allowed a better purification of the compound. This oxidant was able to specifically react with sulfur containing amino acids (methionine and cysteine). In this sense, its reactivity with peroxiredoxins (Prx1 and Prx2) was investigated. HOOU reacted fast with Prx1 k = 4.9 × 105 M-1s-1 and Prx2 k = 2.3 × 106 M-1s-1. In addition, HOOU was able to oxidize Prx2 from intact erythrocytes at the same extend as does hydrogen peroxide. Albumin is an important thiol-containing protein to redox homeostasis in plasma. HOOU was able to oxidize albumin with a rate constant of 0.2 × 102 M-1s-1. Another protein with important function in redox homeostasis is thioredoxin (Trx). Trx was oxidized by HOOU with a rate constant of 2.8 × 102 M-1s-1 and was released together with Prx1 and Prx2 from human macrophages cells (THP-1 cell line) that were incubated with HOOU. The release of these proteins is a signal of cellular stress. Thus, HOOU may be involved in the exacerbation of oxidative stress in inflammatory environments. When neutrophil (HL-60 cell line) and macrophages (THP-1 cell line) were incubated with uric acid and Pseudomonas aeruginosa there was a decrease in hypochlorous acid (HOCl) production because of the competition between chloride and uric acid by myeloperoxidase. It decreased HOCl and impaired the microbicidal activity of the cells, showing that HOOU does not contribute in bacteria clearance. Therefore, the oxidation of uric acid to urate hydroperoxide impairs microbicidal activity and oxidizes thiol-proteins in inflammatory cells contributing to a pro-oxidant status. In this context, the antioxidant role of uric acid in inflammatory response should be reviwed.
|
118 |
Papel de selenoproteínas na neurotoxicidade induzida por metilmercúrio, em camundongos, e potencial bioinseticida de uma alga da Antártica (Prasiola crispa) em modelo de Drosophila melanogasterZemolin, Ana Paula Pegoraro 20 August 2012 (has links)
Submitted by Diego Santos (diegosantos@unipampa.edu.br) on 2015-04-10T11:49:39Z
No. of bitstreams: 1
111010003.pdf: 1306168 bytes, checksum: 65e96979f53ded7b91f629af13358a87 (MD5) / Made available in DSpace on 2015-04-10T11:49:41Z (GMT). No. of bitstreams: 1
111010003.pdf: 1306168 bytes, checksum: 65e96979f53ded7b91f629af13358a87 (MD5)
Previous issue date: 2012-08-20 / O metilmercúrio (MeHg) é um agente tóxico que causa importantes prejuízos à
saúde humana e ambiental. Parte desses efeitos está relacionado a sua capacidade
de induzir estresse oxidativo. Os mecanismos precisos pelos quais o MeHg leva ao
estresse oxidativo ainda não estão bem esclarecidos. Dados na literatura apontam
para a participação de selenoproteínas como a glutationa peroxidase e a tioredoxina
redutase neste processo. Neste estudo, buscou-se investigar o papel de isoformas
de glutationa peroxidase (GPx1 e GPx4) e da tioredoxina redutase (TrxR1) na
neurotoxicidade induzida por MeHg em camundongos, focando na atividade e
expressão destas proteínas. Nossos resultados mostraram, que o tratamento de
camundongos Swiss machos adultos com MeHg (40 mg/L na água de beber)
durante 21 dias causa uma diminuição significativa na atividade das enzimas GPx e
TrxR no córtex e cerebelo dos animais tratados, em comparação com os controles.
Observou-se também uma significativa redução na expressão de GPx1, GPx4 e
TrxR1 no cerebelo dos animais tratados, enquanto que no córtex apenas GPx4 e
TrxR1 foram afetadas. Concomitantemente a estes resultados, observou-se um
aumento significativo na atividade das enzimas antioxidantes SOD, CAT, GR e GST
no cerebelo e da CAT no cortex. A expressão de HSP70 foi significativamente
aumentada no cerebelo dos animais tratados. Estes dados denotam uma clara
resposta celular antioxidante frente aos efeitos tóxicos do MeHg em nosso modelo,
reforçando dados da literatura que indicam o estresse oxidativo como um importante
mecanismos na neurotoxicidade induzida por este organometal. Além disso, nossos
resultados apontam para isoformas de glutationa peroxidase e tioredoxina redutase
como importantes alvos moleculares do MeHg e, ao menos, em nosso
conhecimento, este é o primeiro estudo demonstrando o papel da GPx4 na
neurotoxicidade induzida por este agente tóxico ambiental.
Outro objetivo deste trabalho foi investigar os efeitos biológicos do extrato da alga
Prasiola crispa(PcE), oriunda do continente Antártico, nos modelos de Drosophila
melanogaster e Nauphoeta cinérea. Organismos adaptados a ambientes extremos
como a Antártica tendem a apresentarem uma constituição única em termos de
metabólitos secundários. Desta forma, estudos que visem à elucidação de efeitos
biológicos de organismos oriundos destas regiões tendem a apresentarem relevância do ponto de vista biotecnológico. Nossos dados apontam para um
potencial biocida de PcE nos modelos de mosca-da-fruta (Drosophila melanogaster)
e barata cinerea (Nauphoeta cinerea), visto que a administração do extrato induziu
toxicidade nos dois modelos. A toxicidade em D. melanogaster foi avaliada como
percentagem de mortalidade, atividade locomotora (geotaxia negativa) e alterações
bioquímicas incluindo atividade acetilcolinesterase (AChE) e marcadores de estresse
oxidativo.Também foi investigada a ação cardiotóxica do extrato no modelo de
coração semi-isolado de barata cinerea. A administração do extrato(2mg/ml) foi feita
por 24 horas e, nas moscas, causou um aumento massivo na mortalidade (aumento
de 7,6 vezes em relação ao controle). Também foi observado um aumento
significativo na atividade locomotora, indicando uma ação neurotóxica do extrato. A
atividade AChE, os níveis de glutationa e formação de hidroperóxido manteve-se
inalterada. A atividade da glutationa S-transferase aumentou significativamente após
a administração de PcE, já a atividade da catalase diminuiu significativamente em
moscas que receberam o extrato. No modelo de coração semi-isolado de barata, foi
observado uma diminuição da freqüência cardíaca. A incubação do extrato com
DTNB, um forte agente oxidante, bloqueou significativamente o efeito cardiotóxico
do extrato, sugerindo que compostos redutores podem ser responsáveis pelo efeito
observado. Desta forma, este estudo demonstrou os efeitos tóxicos de PcE, em dois
modelos de inseto, sugerindo seu potencial como bioinseticida. Os mecanismos
precisos relacionados a este efeito ainda necessitam de esclarecimentos, entretanto,
alterações em sistemas antioxidantes vitais podem estar envolvidos. / Methylmercury (MeHg) is a toxic agent that causes severe damage to human health and the environment. These effects are related to its ability to induce oxidative stress. The precise mechanisms by which MeHg leads to oxidative stress are not well understood. Data from literature point to the involvement of selenoproteins such as
glutathione peroxidase and thioredoxin reductase in this process. In this study, we sought to investigate the role of isoforms of glutathione peroxidase (GPx4 and GPx1) and thioredoxin reductase (TrxR1) in MeHg-induced neurotoxicity in mice, focusing on activity and expression of these proteins. Our results showed that treatment of adult male mice Swiss with MeHg (40 mg / L in drinking water) for 21 days causes a significant decrease in GPx and TrxR enzyme activity in the cerebral cortex and cerebellum of treated animals when compared to controls. We also observed a significant reduction in the expression of GPx1, GPx4 and TrxR1 in the cerebellum of the treated animals, whereas in the cortex only GPx4 and TrxR1 are affected..In parallel, we observed a significant increase in antioxidant enzymes SOD, CAT, GR and GST in the cerebellum and CAT in cortex. HSP70 expression was significantly increased in the cerebellum of the treated animals. These results show a clear antioxidant cell response against the toxic effects of MeHg in our model, reinforcing the literature data indicating oxidative stress as an important mechanism in the neurotoxicity induced by this organometal. Furthermore, our results point to isoforms of glutathione peroxidase and thioredoxin reductase as important molecular targets of MeHg and, at least, to our knowledge, this is the first study demonstrating the role of GPx4 in the neurotoxicity induced by this toxic environmental agent. Another objective of this study was to investigate the biological effects of the extract of the alga Prasiola crispa (PcE), from the Antarctic continent, in the insect models Drosophila melanogaster and Nauphoeta cinerea. Organisms adapted to extreme environments such as Antarctica tend to present a unique composition in terms of secondary metabolites. Thus, studies aimed at elucidating the biological effects of organisms from these areas tend to be relevant in a biotechnological point of view. Our data demonstrates a potential biocide PcE effect in the fruit fly (Drosophila melanogaster) and lobster cockroach (Nauphoeta cinerea), since the administration
of the extract induced toxicity in both models. Toxicity in D. melanogaster was assessed as percentage of mortality, locomotor activity (negative geotaxis) and biochemical measurements including acetylcholinesterase (AChE) and markers of oxidative stress. We also investigated the cardiotoxic action of the extract in a cockroach semi-isolated heart model. The administration of the extract (2 mg / ml) for
24 hours toflies, caused a massive increase in mortality (7.6-fold increase compared to control). We also observed a significant increase in locomotor activity, indicating a neurotoxic action of the extract. AChE activity, glutathione levels and the formation of hydroperoxide remained unchanged. The activity of glutathione S-transferase significantly increased after administration of PcE while catalase activity was significantly decreased in flies that received the extract. A significant decrease in heart rate in the cockroach semi-isolated heart model was observed after PcE administration. The incubation of the extract with DTNB, a strong oxidizing agent, significantly blocked the cardiotoxic effect of the extract, suggesting that reducing
compounds may be responsible for the observed effect. Thus, this study demonstrated, for the first time, the toxic effects of PcE, in two insect models, suggesting its potential as a bioinsecticide. The precise mechanisms related to this effect still need clarification, however, changes in vital antioxidant systems may be
involved.
|
119 |
Functional redox compartmentation of GSH in the yeast Saccharomyces cerevisiae / Compartimentalisation du glutathion dans les cellules de levure S. cerevisiae et de ses conséquences fonctionnellesIgbaria, Aeid 23 September 2011 (has links)
L'oxydation des résidus cystéines est une modification biochimique très répandue survenant dans tous les compartiments des cellules eucaryotes. Ce phénomène sert le repliement oxydatif des protéines dans le réticulum endoplasmique (RE), l'importation de protéines dans l'espace intermembranaire de la mitochondrie (IMS). De plus, il a un rôle régulateur dans la matrice mitochondriale et dans le cytosol où il contrôle l’activité des enzymes et des protéines de signalisation et de régulation. Dans tous ces procédés, la réversibilité de l'oxydation des résidus Cys est une caractéristique essentielle. Deux systèmes oxydoréductase puissants existent : les voies de glutathion (GSH) et la thiorédoxine ; ils catalysent la réduction des ponts disulfure, et contrôlent la plupart des processus cellulaires thiol-redox dépendant. Cependant, en dépit d'énormes connaissances portant sur leur enzymologie, peu est connu sur les caractéristiques physiologiques de ces systèmes chez les eucaryotes. Pour déterminer l'importance physiologique de ces systèmes et indiquer lequel est à la base de l'exigence du GSH pour la viabilité, nous avons effectué une analyse complète des cellules de levure épuisée ou contenant des niveaux toxiques de GSH. Les deux conditions déclenchent une réponse « iron-starvation-like » et une altération de l'activité des enzymes d’assemblage des centres fer-soufre (Iron sulfure cluster : ISC) extra-mitochondriales. Cependant, elles n’ont pas d'impact sur l’entretien thiol redox, à l’exception des niveaux élevés de glutathion qui ont altéré le repliement oxydatif des protéines dans le reticulum endoplasmique. Alors que le fer sauve partiellement la maturation des ISC et les défauts de croissance des cellules appauvries eh GSH, des expériences génétiques ont indiqué que, contrairement à la thiorédoxine, le glutathion ne peut pas assurer par lui-même les fonctions thiol-redox de la cellule. Nous proposons que le glutathion soit essentiel par son exigence dans l’assemblage des centres fer-soufre, mais ne serve comme backup que pour maintenir l’état thiol-redox de la cellule. Des niveaux physiologiques élevés de GSH sont ainsi destinés à isoler sa fonction dans le métabolisme du fer des variations de sa concentration pendant le stress redox, ce qui constitue un modèle contestant la vision traditionnelle du GSH comme acteur primordial du contrôle thiol-redox cytosolique.Nos données préliminaires sur la distribution de GSH dans les cellules recueillies par lasurveillance de l'état redox de rxYFP ciblée pour différents compartiments cellulaires (RE,Matrice, cytosol et IMS) dans les cellules HGT1 indiquent un transport spécifique du GSH vers le RE et l'exportation de GSSG de ce compartiment. Nous avons pu caractériser deuxtransporteurs ABC dont la suppression modifie le RE plus oxydant et entraîne une accumulation de GSSG par rapport aux cellules sauvages. Ces données ont été confirmées par le suivi de l'état redox de PDI1 et ERO1 (WT et hyper active). Elles suggèrent un rôle de ces transporteurs dans l'exportation du GSSG du la RE, et que le flux de GSH entre les différents compartiments est très régulé. / Cys residue oxidation is a widespread biochemical modification occurring in all eukaryotic cells compartments. It serves oxidative protein folding in the endoplasmic reticulum (ER), protein import in the intermembrane space of mitochondria (IMS), and it has a regulatory role in the mitochondrial matrix and in the cytosol where it controls enzymes and signaling regulatory proteins activity. In all these processes, reversibility of Cys residue oxidation is a crucial feature. Two potent oxidoreductase systems, the glutathione (GSH) and thioredoxin pathways, catalyze disulfide bond reduction, and presumably control most thiol-redox-dependent cellular processes. However, despite tremendous knowledge of their enzymology, little is known about the physiological features of these systems in eukaryotes. To determine the physiologic importance of these functions and sort out which of them accounts for the GSH requirement for viability, we performed a comprehensive analysis of yeast cells depleted of or containing toxic levels of GSH. Both conditions triggered an intense iron-starvation-like response and impaired the activity of extra-mitochondrial ISC enzymes, but did not impact thiol-redox maintenance, except high glutathione levels that altered oxidative protein folding in the endoplasmic reticulum. While iron partially rescued the ISC maturation and growth defects of GSH-depleted cells, genetic experiments indicated that unlike thioredoxin, glutathione could not support by itself the thiolredox duties of the cell. We propose that glutathione is essential by its requirement in ISC assembly but only serves as a thioredoxin back up in cytosolic thiol-redox maintenance. Glutathione high physiologic levels are thus meant to insulate its function in iron metabolism from variations of its concentration during redox stresses, a model challenging the traditional view of it as prime actor in cytosolic thiol-redox control.Our preliminary data on the distribution of GSH inside cells collected by monitoring the redox state of rxYFP targeted to different cell compartments (ER, Matrix, Cytosol and IMS) in HGT1 cells indicate a specific transport of GSH into the ER and export of GSSG out of it. We were able to characterize two ABC transporters on which their deletion modify the redox state of the ER to more oxidizing and result in accumulation of higher GSSG content compared to WT. These data were confirmed by looking to the redox state of the PDI1 and ERO1 (WT and hyper active), all together suggest a role of these transporters in GSSG export from the ER, and that GSH flux between the different compartments is highly regulated.
|
120 |
Redox-regulation of starch and lipid synthesis in leavesKolbe, Anna January 2005 (has links)
Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves.
<br><br>
Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state.
<br><br>
Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content.
<br><br>
In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels. / Es ist bereits seit längerem bekannt, dass viele Enzyme des Calvinzyklus, des oxidativen Pentosephosphatwegs, des NAD(P)H-Exports und der ATP-Synthese durch post-translationale Redox-Modifikation in Antwort auf Licht reguliert werden. In der vorliegenden Arbeit sollte untersucht werden, ob ein ähnlicher Mechanismus auch die Kohlenstoffspeicherung in Blättern reguliert.
<br><br>
Vorangegangene Studien mit Kartoffelknollen zeigten, dass das Schlüsselenzym der Stärkesynthese ADP-Glukose-Pyrophosphorylase (AGPase) durch die Bildung einer Disulfidbrücke zwischen den zwei kleinen Untereinheiten (AGPB) des tetrameren Proteins inaktiviert wird, die Bedeutung dieses Mechanismus für die Stärkesynthese in Blättern blieb jedoch bislang ungeklärt. Die vorliegenden Arbeiten zeigen, das AGPase in Erbsen-, Kartoffel- und Arabidopsis-Blättern über post-translationale Redox-Modifikation in Antwort auf Tag-Nacht Änderungen reguliert wird. Dies erfolgt über ein Licht-abhängiges Signal, da, erstens, AGPB in isolierten Chloroplasten durch Belichtung sehr schnell von Dimer zu Monomer umgewandelt wird und, zweitens, ein Abdunkeln der Blätter zu einer schnellen Umwandlung von AGPB von Monomer zu Dimer führt. Die Monomerisierung von AGPB ging mit Änderungen in den kinetischen Eigenschaften des Enzyms einher, die zu einer Aktivierung führten. Studien mit Extrakten aus Erbsenchloroplasten zeigten, dass die AGPase-Redoxaktivierung in-vitro durch die Thioredoxine f und m aus Spinat vermittelt wird. In einem weiteren experimentellen Ansatz konnte gezeigt werden, dass auch Zucker zu Redox-Aktivierung der AGPase und erhöhter Stärkesynthese in Blättern führen, und dass diese unabhängig von Licht wirken. Externe Zugabe von Zuckern wie Saccharose oder Trehalose an Arabidopsis-Blätter im Dunkeln führten zu Monomerisierung von AGPB und einer Erhöhung der Stärkesyntheserate / während die Spiegel des allosterischen Aktivators 3PGA unverändert blieben und keine Änderungen im NADPH/NADP+-Verhältnis auftraten. Experimente mit transgenen Arabidopsis-Pflanzen mit veränderten Spiegeln des Vorläufers der Trehalosesynthese, Trehalose-6-phosphat (T6P), zeigten, dass T6P und nicht Trehalose zu Redox-Aktivierung von AGPase führt. Expression einer E. coli T6P synthase (TPS) im Zytosol führte zu erhöhter Redox-Aktivierung von AGPase und erhöhter Stäreksynthese in Blättern, während die Expression einer T6P-Phosphatase (TPP) gegenteilige Änderungen bewirkte. Diese Auswirkungen erfolgten unabhängig von Änderungen in den Spiegeln von Zuckern und Zuckerphosphaten oder im NADPH/NADP+-Verhältnis. Externe Zugabe von Saccharose führte zu Monomerisierung von AGPB in Wildtyp und TPS exprimierenden Blättern, während diese Antwort in TPP exprimierenden Blättern stark abgeschwächt war. Dies zeigt, dass T6P eine wesentliche Komponente darstellt, die die Redox-Aktivierung der AGPase in Antwort auf Saccharose vermittelt. T6P wurde auch für 15 min direkt an intakte, isolierte Erbsenchloroplasten gefüttert. T6P Konzentrationen im Bereich von 100 µM bis 10 mM führten zu einem signifikanten und spezifischen Anstieg der AGPB-Monomersierung und der AGPase Aktivität. Dies zeigt, dass T6P auch ohne zytosolische Elemente die Redox-Aktivierung der AGPase stimuliert und somit ein Signal zwischen Zytosol und Plastid darstellt. Diese Antwort erfolgte ohne Änderungen im NADPH/NADP+-Verhältnis, was zeigt, dass T6P eher den Redox-Transfer zu AGPase als den Redoxzustand des Chloroplasten moduliert.
<br><br>
Acetyl-CoA-Carboxylase (ACCase) ist als Schlüsselenzym der Fettsäure- und Lipidsynthese in Pflanzen bekannt. Zu Beginn der vorliegenden Arbeit lagen hauptsächlich in-vitro Befunde vor, die zeigten, dass ACCase durch DTT und thioredoxine f und m über Redox-Modulation reguliert wird. In der Arbeit sollte daher die in-vivo Relevanz dieses Mechanismus für die Regulation der Lipidsynthese in Blättern untersucht werden. ACCase zeigte einen höheren Redox-Aktivierungszustand in Arabidopsis-Blätter, die während des Tages im Vergleich zur Nacht geerntet wurden. Die Redox-Aktivierung der ACCase wurde von Änderungen in den kinetischen Eigenschaften begleitet und führte zu einer erhöhten Affinität des Enzymes gegenüber Acetyl-CoA als Substrat.
<br><br>
In weiteren Versuchen wurde sowohl DTT als auch Saccharose an Blätter gefüttert, und beide Behandlungen führten zu Redox-Aktivierung von ACCase, was mit erhöhten Lipidsynthesraten und einem Rückgang des Acetyl-CoA-Spiegels einherging.
|
Page generated in 0.037 seconds