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Dynamic Rerouting of the Carbohydrate Flux Is Key to Counteracting BioMed Central Open Access Research article Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress Markus Ralser*, Mirjam M Wamelink†, Axel Kowald*¶, Birgit Gerisch*, Gino Heeren§, Eduard A Struys†, Edda Klipp*, Cornelis Jakobs†, Michael Breitenbach§, Hans Lehrach* and Sylvia Krobitsch* Addresses: *Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. †Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. §Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. ¶Current address: Medical Proteome Center, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. Correspondence: Markus Ralser. Email: [email protected]; Sylvia Krobitsch. Email: [email protected] Published: 21 December 2007 Received: 21 May 2007 Revised: 7 August 2007 Journal of Biology 2007, 6:10 (doi:10.1186/jbiol61) Accepted: 12 October 2007 The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/6/4/10 © 2007 Ralser et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role. Results: We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in Caenorhabditis elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux. Conclusions: The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions. Journal of Biology 2007, 6:10 10.2 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Background observations above suggest that alterations in the carbohy- Reactive oxygen species (ROS) cause damage to cellular drate metabolism could be central for cellular protection processes in all living organisms and contribute to a against ROS and, moreover, that cells reroute the carbohy- number of human disorders such as cancer, cardiovascular drate flux from glycolysis to the PPP to counteract perturba- diseases, stroke, and late-onset neurodegenerative disorders, tions in the cytoplasmic redox state. However, direct and to the aging process itself. To cope with the fatal cellular evidence for this hypothesis is missing so far. By combining consequences triggered by ROS, eukaryotic cells have genetic and quantitative metabolite analyses along with in evolved a number of defense and repair mechanisms, which silico modeling, we present the first direct proof that eukary- are based on enzymatic as well as non-enzymatic processes otic cells indeed actively reroute the metabolic flux from and appear to be highly conserved from unicellular to glycolysis to the PPP as an immediate and protective multicellular eukaryotes. In bacteria and yeast, these anti- response to counteract oxidative stress. oxidant defense mechanisms are partially induced on the basis of changes in global gene expression [1,2]. However, a recent study analyzing a number of genetic and environ- mental perturbations in Escherichia coli demonstrated that Results the changes in the transcriptome and proteome are unex- Reduced intracellular TPI concentration results in pectedly small [3]. Moreover, the transcription of genes enhanced oxidant resistance of Saccharomyces encoding enzymes capable of neutralizing ROS is not gener- cerevisiae and Caenorhabditis elegans ally increased in mammalian cells that are subjected to We reported earlier that a change of the amino acid oxidative stress [4]. isoleucine to valine at position 170 in the human TPI protein (TPIIle170Val) causes a reduction of about 70% in the In all organisms studied, however, treatment with oxidants enzyme’s catalytic activity [11]. Interestingly, we discovered prompts immediate de novo post-translational modifica- that yeast cells expressing this human TPI variant exhibit tions of a number of proteins, probably affecting their local- increased resistance to the oxidant diamide (N,N,N’,N’- ization and functionality. One of the key targets of those tetramethylazodicarboxamide, Chemical Abstracts Service processes is the glycolytic enzyme glyceraldehyde-3-phos- (CAS) No. 10465-78-8) compared with isogenic yeast cells phate dehydrogenase (GAPDH), which catalyzes the expressing wild-type human TPI, indicating that low TPI reversible oxidative phosphorylation of glyceraldehyde-3- activity confers resistance to specific conditions of oxidative phosphate (gly3p) to 1,3-bisphosphoglycerate. Remarkably, stress. The synthetic reagent diamide is known to oxidize in response to various oxidant treatments this enzyme is cellular thiols, especially protein-integrated cysteines [19], inactivated and transported into the nucleus of the cell, and provoking a rapid decrease in cellular glutathione and has been found S-nitrosylated, S-thiolated, S-glutathiony- hence causing oxidative stress. To dissect the underlying lated, carbonylated and ADP-ribosylated in numerous cell mechanism, we first analyzed whether decreasing the types and organisms under these conditions [5-10]. expression level of wild-type human TPI would result in a Recently, we discovered that yeast cells with reduced cat- similar phenotype. For this, we generated plasmids for the alytic activity of another key glycolytic enzyme, triose- expression of wild-type human TPI under the control of phosphate isomerase (TPI), are highly resistant to the established yeast promoters of different strengths, namely oxidant diamide [11]. This essential enzyme precedes the CYC1, TEF1 and GPD1 promoters [20]. Subsequently, GAPDH in glycolysis, catalyzing the interconversion of di- the ∆tpi1 strain MR100, which is deleted for the yeast TPI1 hydroxyacetone phosphate (dhap) and gly3p, the substrate gene and is inviable on medium containing glucose as sole of GAPDH, and a reduction in its activity results in an ele- carbon source, was transformed with the respective plas- vated cellular dhap concentration [12-14]. In this light, it is mids along with control plasmids encoding yeast TPIIle170Val remarkable that the expression of a subset of glycolytic pro- or yeast TPI. Single colonies were selected and the intracel- teins and proteins implicated in related pathways is repres- lular TPI concentration of plate-grown yeast cells was ana- sed, while the expression of a few enzymes involved in the lyzed (Figure 1a, left panel). As expected, yeast cells pentose phosphate pathway (PPP), which is directly con- expressing the different TPI proteins under the strong GPD1 nected to the glycolytic pathway, is induced under oxidative promoter had a higher TPI concentration compared with stress conditions [1]. Furthermore, enhanced activity of the cells in which the expression was controlled by the interme- PPP has been observed in neonatal rat cardiomyocytes and diate TEF1 or the weak CYC1 promoter. Next, we spotted in human epithelial cells under oxidative stress conditions the respective yeast cells onto medium supplemented with [15,16]. Enzymes of the PPP are crucial for maintaining the differing diamide concentrations. As shown in Figure 1a cytoplasmic NADPH concentration, which provides the (right panel), yeast cells expressing human TPI under the redox power for known antioxidant systems [17,18]. The control of the weakest promoter used, the CYC1 promoter, Journal of Biology 2007, 6:10 http://jbiol.com/content/6/4/10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. 10.3 (a) lle170Val -TPI -yeast TPI-TPI -TPI -TPI GPD -yeast TPI pr pr pr pr pr pr GPD GPD GPD TEF CYC GPDpr -TPIlle170Val TPI GPDpr -TPI TEFpr -TPI G6PDH CYCpr -TPI Without diamide 1.4 mM diamide 1.6 mM diamide 1.8 mM diamide (b) Juglone Diamide RNAi 100 100 Control tpi-1 90 90 80 80 TPI-1 70 70 Wild-type 60 60 50 50 tpi-1 RNAi DAF-21 40 40 30 30 daf-2 (e1370) 20 20 10 10 0 0 Animals alive (percentage) alive Animals 0 1 2 3 4 5 6 7 8 (percentage) alive Animals 0123456789 Time (h) Time (h) Figure 1 Reduced triosephosphate isomerase (TPI) activity increases oxidant resistance of S. cerevisiae and C. elegans. (a) The left panel shows a Western blot analysis of yeast cells expressing wild-type human TPI under the control of promoters of different strengths: GPD1 (GPDpr), TEF1 (TEFpr), and CYC1 (CYCpr). Yeast cells expressing human TPIIle170Val or yeast TPI under the control of the strong
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