Glutathione synthesis is essential for mouse development but not for cell growth in culture

Shi, Zheng Zheng; Osei-Frimpong, Joseph; Kala, Geeta; Kala, Subbarao V.; Barrios, Roberto; Habib, Geetha M.; Lukin, Dana J.; Danney, Christopher M.; Matzuk, Martin M.; Lieberman, Michael W.

doi:10.1073/pnas.97.10.5101

Cited by 255 publications

(201 citation statements)

References 41 publications

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“…Mice bearing mutations of genes that are involved in regulating cellular redox status also show early lethality. For example, mice with mutations in genes such as thioredoxin, selenocysteine tRNA, Ref1, and the Gclc result in developmental failure at implantation (41)(42)(43)(44)(45). Thioredoxin is a selenoprotein that regulates expression of various genes through the redox factor 1 protein (Ref1).…”

Section: Discussionmentioning

confidence: 99%

Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress

Leung

Kwong

Hou

et al. 2003

Journal of Biological Chemistry

284

243

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Nrf1 and Nrf2 are members of the CNC family of bZIP transcription factors that exhibit structural similarities, and they are co-expressed in a wide range of tissues during development. Nrf2 has been shown to be dispensable for growth and development in mice. Nrf2-deficient mice, however, are impaired in oxidative stress defense. We previously showed that loss of Nrf1 function in mice results late gestational embryonic lethality. To determine whether Nrf1 and Nrf2 have overlapping functions during early development and in the oxidative stress response, we generated mice that are deficient in both Nrf1 and Nrf2. In contrast to the late embryonic lethality in Nrf1 mutants, compound Nrf1, Nrf2 mutants die early between embryonic days 9 and 10 and exhibit extensive apoptosis that is not observed in the single mutants. Loss of Nrf1 and Nrf2 leads to marked oxidative stress in cells that is indicated by elevated intracellular reactive oxygen species levels and cell death that is reversed by culturing under reduced oxygen tension or the addition of antioxidants. Compound mutant cells also show increased levels of p53 and induction of Noxa, a death effector p53 target gene, suggesting that cell death is potentially mediated by reactive oxygen species activation of p53. Moreover, we show that expression of genes related to antioxidant defense is severely impaired in compound mutant cells compared with single mutant cells. Together, these findings indicate that the functions of Nrf1 and Nrf2 overlap during early development and to a large extent in regulating antioxidant gene expression in cells.Reactive oxygen species (ROS) 1 are generated during aerobic respiration and normal metabolic processes, and they are also byproducts of metabolism of a wide range of environmental agents (1). High levels of ROS are detrimental to the cell, since they react readily with intracellular molecules, causing cell injury and death (1). Cells are equipped with a variety of defense mechanisms that work in parallel or in sequence to minimize ROS levels. These defenses include enzymes that are involved in ROS metabolism and biotransformation of xenobiotics (2). Examples of these enzymes include superoxide dismutases, glutathione peroxidases, thioredoxins, and heme-oxygenases as well as phase 2 enzymes, such as glutathione S-transferases. In addition to enzymatic defenses, cells are also equipped with molecules such as glutathione, metallothioneins, and ferritins that scavenge ROS and metal ions. Basal and inducible expression of a number of these antioxidant defense genes are mediated in part by a cis-acting DNA element known as the antioxidant response element (3-6). Activation through the antioxidant response element (ARE) appears to be driven by conditions that promote intracellular oxidative stress (5,7,8). A number of different transcription factors including basic leucine zipper proteins, AP-1, have been shown to bind the ARE (3, 9, 10).Recent studies implicate the CNC subfamily of bZIP proteins in mediating ARE function (11). Members o...

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Section: Discussionmentioning

confidence: 99%

Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress

Leung

Kwong

Hou

et al. 2003

Journal of Biological Chemistry

284

243

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“…Alterations in the cellular redox status have been shown to control embryo development in several systems (Earnshaw and Johnson 1987;Gardiner et al 1998;Shi et al 2000;Stasolla et al 2004a;Belmonte et al 2006). In recent studies on B. napus, Belmonte et al (2006) show that the endogenous glutathione redox status [deWned as the ratio of the concentration of the reduced form (GSH) to the concentration of the oxidized plus reduced forms (GSSG + GSH)] can be easily manipulated in culture through applications of buthionine sulfoximine (BSO).…”

Section: Introductionmentioning

confidence: 99%

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

Stasolla

Belmonte

Tahir

et al. 2008

Planta

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Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively inXuences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript proWles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO aVected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major Xuctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their postembryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARG-ONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle-and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage-and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.

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“…Null mutations in GSH1 result in GSH auxotrophy (14 -16), demonstrating that GSH is essential in yeast. In mammals, GSH is also essential, as demonstrated by the embryonic lethality resulting from disruption of ␥-glutamyl cysteine synthetase and by the inability of blastocysts isolated from such knock-out strains to grow, except in the presence of GSH or the GSH substitute N-acetyl cysteine (17). The reason for the essential requirement of GSH in eukaryotes is not known.…”

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confidence: 99%

A Genetic Investigation of the Essential Role of Glutathione

Spector

Labarre

Toledano

2001

Journal of Biological Chemistry

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In an attempt to elucidate the essential function of glutathione in Saccharomyces cerevisiae, we searched for suppressors of the GSH auxotrophy of ⌬gsh1, a strain lacking the rate-limiting enzyme of glutathione biosynthesis. We found that specific mutations of PRO2, the second enzyme in proline biosynthesis, permitted the growth of ⌬gsh1 in the absence of exogenous GSH. The suppression mechanism by alleles of PRO2 involved the biosynthesis of a trace amount of glutathione. Deletion of PRO1, the first enzyme of the proline biosynthesis pathway, or PRO2 eliminated the suppression, suggesting that ␥-glutamyl phosphate, the product of Pro1 and the physiological substrate of Pro2, is required as an obligate substrate of suppressor alleles of PRO2 for glutathione synthesis. A mutagenesis of a ⌬gsh1 strain also lacking the proline pathway failed to generate any suppressor mutants under either aerobic or anaerobic conditions, confirming that glutathione is essential in yeast. This essential function is not related to DNA synthesis based on the terminal phenotype of glutathione-depleted cells or to toxic accumulation of non-native protein disulfides. Analysis of the suppressor strain demonstrates that normal glutathione levels are required for the tolerance to oxidants under acute, but not chronic stress conditions. Glutathione (GSH) is a broadly conserved tripeptide with a highly reactive thiol and a very low redox potential of about Ϫ240 to Ϫ250 mV. Its elevated concentration, up to 10 mM, and the fact that its reduced state is efficiently maintained by NADPH-dependent glutathione reductase (reviewed in Refs. 1-3) confers to this small molecule the properties of a cellular redox buffer. As a redox buffer, GSH is thought to be a major determinant in maintaining a reducing cellular thiol-disulfide balance. GSH is also important as an electron donor for several enzymes that have a reducing step in their catalytic cycle, such as ribonucleotide reductase (4). In these situations, electrons from GSH are generally transduced to their substrates through glutaredoxins. GSH may protect protein sulfhydryls from irreversible oxidation by glutathionylation (5, 6). It also participates in the detoxification of peroxides, either directly or indirectly as a cofactor of glutathione peroxidase (1,7,8) and of several chemicals such as cadmium (9, 10).In Saccharomyces cerevisiae and in probably all other GSHcontaining organisms, GSH is synthesized in two steps beginning with the action of ␥-glutamyl cysteine synthetase (GSH1) (11, 12), which catalyzes the condensation of glutamic acid to cysteine, in the rate-limiting step of this biosynthetic pathway. The product of this reaction is ␥-glutamylcysteine, which is combined with glycine through the action of glutathione synthase (GSH2) (13) to form GSH. Null mutations in GSH1 result in GSH auxotrophy (14 -16), demonstrating that GSH is essential in yeast. In mammals, GSH is also essential, as demonstrated by the embryonic lethality resulting from disruption of ␥-glutamyl cysteine synthetase ...

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Glutathione synthesis is essential for mouse development but not for cell growth in culture

Cited by 255 publications

References 41 publications

Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress

Deficiency of the Nrf1 and Nrf2 Transcription Factors Results in Early Embryonic Lethality and Severe Oxidative Stress

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

A Genetic Investigation of the Essential Role of Glutathione

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