RESUMEN
IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.
Asunto(s)
Glioma/metabolismo , Ácido Glutámico/biosíntesis , Transaminasas/fisiología , Línea Celular Tumoral , Glioma/fisiopatología , Ácido Glutámico/efectos de los fármacos , Glutaratos/metabolismo , Glutaratos/farmacología , Homeostasis/efectos de los fármacos , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/fisiología , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/fisiología , Mutación , Oxidación-Reducción/efectos de los fármacos , Proteínas Gestacionales/genética , Proteínas Gestacionales/fisiología , Transaminasas/antagonistas & inhibidores , Transaminasas/genéticaRESUMEN
NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. In animal, yeast and bacteria, the NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defence mechanisms. In plant cells, there is little information on the antioxidant properties of NADP-ICDH. Using a pea cDNA lambdagt11 library, the full-length cDNA of a NADP-ICDH was obtained. In pea leaves, the analyses of activity, protein and transcript expression of NADP-ICDH under six different abiotic stress conditions (CL, continuous light, HLI, high light intensity, D, continuous dark, LT, low-temperature HT, high-temperature and W, mechanical wounding) revealed a differential regulation at transcriptional and post-translational level depending on the abiotic stress. The activity and protein expression of NADP-ICDH and catalase increased only under HLI but the NADP-ICDH transcripts were up-regulated by cold stress (70%) and W (40%). Under the same conditions, the transcript analysis of glutathione reductase (GR), monodehydroascorbate reductase (MDAR) and ascorbate peroxidase (APX), key components of the antioxidative ascorbate-glutathione cycle, showed similar inductions. These data indicate that in pea plants the cytosolic NADP-ICDH shows a differential response, at mRNA and activity level, depending on the type of abiotic stress and suggests that this dehydrogenase could have a protective antioxidant role against certain environmental stresses in plants.
Asunto(s)
Citosol/enzimología , Isocitrato Deshidrogenasa/genética , Pisum sativum/enzimología , Proteínas de Plantas/genética , Ascorbato Peroxidasas , Catalasa/análisis , Frío , ADN Complementario/genética , ADN de Plantas/genética , Relación Dosis-Respuesta en la Radiación , Inducción Enzimática/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glutatión Reductasa/biosíntesis , Glutatión Reductasa/genética , Calor , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/fisiología , Luz , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/biosíntesis , NADH NADPH Oxidorreductasas/genética , Pisum sativum/genética , Pisum sativum/efectos de la radiación , Peroxidasas/biosíntesis , Peroxidasas/genética , Hojas de la Planta/enzimología , Hojas de la Planta/efectos de la radiación , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , ARN Mensajero/biosíntesis , ARN de Planta/biosíntesis , Estrés MecánicoRESUMEN
NAD-dependent isocitrate dehydrogenase (IDH) is a Krebs cycle enzyme situated in mitochondria. In Arabidopsis thaliana, five genes encode functional IDH subunits that can be classed into two groups based on gene structure and subunit amino acid sequence. Arabidopsis contains two 'catalytic' and three 'regulatory' subunits according to their homology with yeast IDH. To date, an active IDH is believed to be heteromeric, containing at least one of each subunit type. This was verified in Arabidopsis by the complementation of yeast IDH mutants with the different Arabidopsis IDH-encoding cDNAs. Indeed, a single 'catalytic' and 'regulatory' subunit was sufficient to restore acetate growth of the yeast IDH double mutant. To gain information on possible IDH subunit interactions in planta, Arabidopsis IDH gene expression was analysed by Northern blot, PCR on cDNA libraries, in silico and in 'promoter'-reporter gene transgenic plants. Four of the IDH genes were expressed in all plant organs tested, while one gene (At4g35650) was not expressed in vegetative organs but was mainly expressed in the pollen. In leaves, the IDH genes were highly expressed in the veins, and to a lesser extent in mesophyll cells. The data are discussed with respect to IDH in other plant species.