Folding of the four domains and dimerization are impaired by the Gly446.fwdarw.Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs

Nordhoff, Axel; Us, Bücheler; Werner, Dieter; Rh, Schirmer

doi:10.1021/bi00066a029

Cited by 114 publications

(102 citation statements)

References 42 publications

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“…Recombinant human GR was prepared as described in ref. 21. To measure GR activity, 0.5 g of plant material was mixed with 2 ml of 0.1 M Tris, 1 mM EDTA, and 7.5% wt͞vol polyvinylpyrrolidone (pH 7.8).…”

Section: Methodsmentioning

confidence: 99%

“…After centrifugation, protein was determined in the supernatant according to Bradford (22) by using BSA as the standard. GR activity was measured spectrophotometrically (for NADPH, 340 ϭ 6.22 mM Ϫ1 ⅐cm Ϫ1 ) in 47 mM potassium phosphate, 200 mM KCl, and 1 mM EDTA (pH 6.9) at 25°C (21). A baseline was monitored in the presence of plant extract and 100 M NADPH to account for NADPH oxidase activity.…”

Section: Methodsmentioning

confidence: 99%

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The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

Waller

Achatz

Baltruschat

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

1,150

768

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Disease resistance strategies are powerful approaches to sustainable agriculture because they reduce chemical input into the environment. Recently, Piriformospora indica, a plant-root-colonizing basidiomycete fungus, has been discovered in the Indian Thar desert and was shown to provide strong growth-promoting activity during its symbiosis with a broad spectrum of plants [Verma, S. et al. (1998) Mycologia 90, 896 -903]. Here, we report on the potential of P. indica to induce resistance to fungal diseases and tolerance to salt stress in the monocotyledonous plant barley. The beneficial effect on the defense status is detected in distal leaves, demonstrating a systemic induction of resistance by a root-endophytic fungus. The systemically altered ''defense readiness'' is associated with an elevated antioxidative capacity due to an activation of the glutathione-ascorbate cycle and results in an overall increase in grain yield. Because P. indica can be easily propagated in the absence of a host plant, we conclude that the fungus could be exploited to increase disease resistance and yield in crop plants.root endophyte ͉ powdery mildew ͉ symbiosis ͉ ascorbate ͉ glutathione

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

Waller

Achatz

Baltruschat

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

1,150

768

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“…Recombinant human glutathione reductase was prepared as described previously (Nordhoff et al, 1993). Becf heart glutamate dehydrogenase was purchased froin Boehringer Mannheim.…”

Section: Methodsmentioning

confidence: 99%

“…Becf heart glutamate dehydrogenase was purchased froin Boehringer Mannheim. The immunoadsorption gel was prepared by covalently coupling polyclonal rabbit antibodies against human glutathione reductase to protein-A -Sepharose CL-4B (Nordhoff et al, 1993). Ado(2',5')P,-Sephilro~e, Q-Sepharose, and a Superose 12 column were obtained from Pharmacia, Ccntricon 10 concentrators were from Amicon, bicinchoninic acid and the micro-bicinchoninic acid protein assay kit were from Pierce, the quick silver protein stain kit was from Amersham, and cellulose acetate sheets (Cellogel) were from Biotec Fischer.…”

Section: Methodsmentioning

confidence: 99%

Glutathione Reductase and Glutamate Dehydrogenase of Plasmodium Falciparum, The Causative Agent of Tropical Malaria

Krauth‐Siegel

Müller

Lottspeich

et al. 1996

European Journal of Biochemistry

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The use of glutathione reductase inhibitors in chemotherapy is the raison d'&tre for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum : glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N-terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first /j strand and the diphosphate-fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of I? ,fulcipurunz glutathione reductase as a drug target are discussed.

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“…Another reason might be the synthesis of monomers that do not dimerize co-translationally and are therefore degraded rapidly by the bacterial cell. It has been shown previously that monomeric mouse glutathione reductase is degraded rapidly by bacterial cells (22). The generation of E. coli glutathione reductase hybrid protein species resulted in high yields of proteins, and there was no mention about a differential expression pattern concerning the different protein species that were generated (23).…”

Section: Expression and Purification Of Pftrxr Homo-and Heterodimers-mentioning

confidence: 99%

Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

Krnajski

Gilberger

Walter

et al. 2000

Journal of Biological Chemistry

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The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M r and the low M r proteins. The well characterized Escherichia coli TrxR represents a member of the low M r class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M r proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M r TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.Infection with Plasmodium falciparum, the causative agent of malaria tropica, is responsible for 2-3 million deaths per year. The malaria parasite spends part of its developmental life cycle in human erythrocytes where it is challenged with enhanced oxidative stress. Therefore the parasite needs efficient anti-oxidants to protect itself against damages, such as nucleic acid modifications, lipid peroxidation, or oxidation of thiolcontaining proteins, caused by reactive oxygen species. The thioredoxin redox system, composed of the NADPH-dependent homodimeric thioredoxin reductase (TrxR) 1 and the 12-kDa protein thioredoxin (Trx) confers reduction of protein disulfides, ribonucleotide reductase being the most prominent example (1). Apart from this, thioredoxin interacts with a number of transcription factors in prokaryotic and eukaryotic cells, resulting in modified DNA binding activities and altered gene transcription (2). Another important function is the interaction of reduced thioredoxin with thioredoxin-dependent peroxidases, which detoxify reactive oxyg...

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Folding of the four domains and dimerization are impaired by the Gly446.fwdarw.Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs

Cited by 114 publications

References 42 publications

The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield

Glutathione Reductase and Glutamate Dehydrogenase of Plasmodium Falciparum, The Causative Agent of Tropical Malaria

Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

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